Anti-factor IX Padua antibodies

ABSTRACT

Provided herein are anti-Factor IX Padua binding constructs, e.g., antibodies and antigen-binding fragments thereof. Related polypeptides, conjugates and kits are also provided. The inventions may be used in methods of detecting Factor IX Padua in a sample.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application is a U.S. National Phase Application under 35U.S.C. § 371 of International Patent Application No. PCT/US2017/032808,filed May 16, 2017 and published on Nov. 23, 2017 as InternationalPublication No. WO 2017/200981A1, which claims priority pursuant to 35U.S.C. § 119(e) to U. S. Provisional Patent Applications Nos.62/337,118, filed May 16, 2016 and 62/340,834, filed May 24, 2016. Theentire contents of these applications are hereby incorporated byreference in their entirety.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: 28,804 byte ACII (Text) file named“50573_SeqListing.txt”; created on May 16, 2016.

BACKGROUND

Gene therapy holds great promise as a future treatment option forhemophilia. In one clinical trial, subjects with severe hemophilia B aretreated with an adeno-associated viral (AAV) vector encoding Factor IX(FIX) Padua, a hyper-functional variant of FIX with a single amino acidsubstitution of Leu for the Arg at position 338 of the mature peptidesequence (or position 384 of the preproprotein sequence). Specificdetection of the transgene product (FIX Padua) would be useful forassessing the success of factor replacement. However, specific detectionof FIX Padua in treated patients remains a challenge, as some patientshave Factor IX cross-reactive material (CRM). Some CRM-positive (CRM+)patients express, for example, wild-type (WT) Factor IX, whichcross-reacts with currently available FIX Padua-binding agents, thusmaking it difficult to determine if FIX Padua is expressed by such CRM+patients.

SUMMARY

Provided herein are binding constructs which specifically recognize FIXPadua (FIXp) without cross-reactivity to wild-type FIX. In exemplaryembodiments, the binding construct is an antibody or an antigen-bindingfragment thereof that binds FIX Padua comprising the amino acid sequenceof SEQ ID NO: 1 and does not bind to a WT Factor IX comprising the aminoacid sequence SEQ ID NO: 2. In exemplary embodiments, the bindingconstruct is a polypeptide comprising an amino acid sequence comprisingeach of SEQ ID NOs: 6-11, optionally, wherein (i) one or more aminoacids are present between each of SEQ ID NOs: 6-11, and/or (ii) thepolypeptide optionally further comprises a FLAG tag comprising DYKDDDDK(SEQ ID NO: 12) and/or a hexa-His tag comprising HHHHHH (SEQ ID NO: 13),optionally, wherein the FLAG tag and/or the hexa-His tag are located atthe C-terminal end of the polypeptide. In exemplary embodiments, thebinding construct is a conjugate comprising an antigen-binding fragmentas described herein conjugated to (i) a constant region of animmunoglobulin heavy chain, (ii) a constant region of an immunoglobulinlight chain, or (iii) both a constant region of an immunoglobulin heavychain and a constant region of an immunoglobulin light chain. Inexemplary embodiments, the binding construct is a conjugate comprisingan antibody or antigen-binding fragment as described herein linked orconjugated to a heterologous moiety. In exemplary aspects, the conjugatecomprises an antibody or antigen-binding fragment as described hereinconjugated to a polymer, a carbohydrate, a lipid, a nucleic acid, anoligonucleotide, an amino acid, peptide, polypeptide, protein, or adetecting agent.

Nucleic acids comprising a nucleotide sequence encoding the antibody,antigen-binding fragment, polypeptide, conjugate, or a fragment thereof,as described herein, are additionally provided. Vectors comprising thenucleic acid and host cells comprising the nucleic acid or vector arefurther provided.

Also provided herein are related kits. In exemplary embodiments, the kitcomprises an antibody, antigen-binding fragment, polypeptide, conjugate,nucleic acid, vector, host cell, as described herein, or a combinationthereof, and, optionally, instructions for use. In exemplary aspects,the kit also comprises a solid support and optionally, the antibody,antigen-binding fragment, polypeptide, or conjugate is pre-coated on thesolid support. In exemplary aspects, the kit also comprises a secondaryantibody which binds to the antibody, antigen-binding fragment,polypeptide, or conjugate provided in the kit.

The invention further provides compositions comprising an antibody orantigen-binding fragment or polypeptide or conjugate, as describedherein, admixed with a biological sample, e.g., a biological sampleobtained from a human. In exemplary aspects, the biological samplecomprises human plasma, or a diluted fraction thereof, and/or humantissue, or cells thereof. In exemplary aspects, the biological samplecomprises human plasma proteins, wherein at least one of the humanplasma proteins is selected from the group consisting of Factor IX,Factor II, and Factor X, and variants thereof. Optionally, thecomposition comprises a detecting agent.

Such binding constructs provided herein are useful in, e.g., detectionmethods that allow for unambiguous or specific detection of FIX Padua insamples, e.g., clinical or preclinical samples comprising, e.g.,wild-type FIX. Accordingly, provided herein are methods of detectingFactor IX Padua comprising the amino acid sequence of SEQ ID NO: 1 in asample obtained from a subject. In exemplary embodiments, the methodcomprises (i) contacting the sample with a binding construct (e.g., anantibody, antigen-binding fragment, polypeptide or conjugate, asdescribed herein) to form a complex, e.g., an immunocomplex, comprisingthe FIXp and the binding construct, and (ii) detecting the complex inthe sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents a schematic of the amino acid sequences of WT FactorIX (SEQ ID NO: 2) and of FIX Padua (SEQ ID NO: 1).

FIG. 2 represents a graph of the binding signals of FIX Padua (leftpanel) or WT Factor IX (right panel) to Ni²⁺ plates coated with 5 μg/mlBC1 (blue bars closest to Z-axis (i.e., to the left)), 1 μg/ml BC1 (redbars right of the blue bars), 0.2 μg/ml BC1 (green bars adjacent to thered bars) or 0.04 μg/ml BC1.

FIG. 3 represents a graph of the binding signals of BC1 (triangles), BC2(diamonds), or BC3 (squares) coated on MaxiSorp plates to the indicatedconcentration of FIX Padua.

FIG. 4 represents a graph of the binding signals of BC1 to the indicatedconcentration of FIX Padua in 5% human plasma solution containing 5μg/ml WT Factor IX without benzamidine.

FIG. 5 represents a graph of the binding signals of BC1 to the indicatedconcentration of FIX Padua in 2% BSA/PBS (diamonds), 5% plasma solutioncontaining 50 mM benzamidine (squares), 10% plasma solution containing50 mM benzamidine (triangles), or 20% plasma solution containing 50 mMbenzamidine (Xs).

FIG. 6 represents a graph of the binding signals of BC1 (squares), BC2(triangles), BC4 (diamonds), or a negative control (Xs) to the indicatedconcentration of FIX Padua in a 20% (v/v) plasma solution comprising WTFactor IX and 50 mM benzamidine.

FIG. 7 represents two graphs of the binding signals of BC1 (left panel)and BC4 (right panel) to the indicated concentration of coagulationfactors: FIX Padua (diamonds); Factor II (squares); and Factor X(triangles).

FIG. 8 represents two graphs of the binding signals of BC5 (left panel)and BC6 (right panel) to the indicated concentrations (μg/ml) of WTFactor IX (diamonds) or to FIX Padua (squares).

FIG. 9 represents a schematic of the components of the ELISA describedin Example 2.

FIG. 10 represents a schematic of the components of the ELISA describedin Example 1.

FIG. 11 represents a graph of concentration-response curves of the FIXPadua ELISA using a sample containing FIX Padua (circles) or a referencehuman plasma sample (diamonds).

FIG. 12 represents a graph of a calibration curve of the FIX Padua ELISAusing six standards of the indicated amounts of FIX Padua (standards D1to D6).

FIG. 13 represents a graph of dilution-response curves of the FIX PaduaELISA using FIX Padua in buffer or in normal plasma or FIX-deficientplasma.

FIG. 14 represents a graph of dilution-response curves of the FIX PaduaELISA using citrated monkey plasma samples.

FIG. 15 represents a graph of the clotting activity and proteinmeasurements of samples of a subject with FIX cross-reactive material(CRM+) after treatment with AAV2/8 viral vector expressing FIX Padua.

FIG. 16 represents a graph of the clotting activity and proteinmeasurements of samples of a second subject with FIX cross-reactivematerial (CRM+) after treatment with AAV2/8 viral vector expressing FIXPadua.

FIG. 17 represents a graph of the clotting activity and proteinmeasurements of samples of a subject without FIX cross-reactive material(CRM+) after treatment with AAV2/8 viral vector expressing FIX Padua.

FIG. 18 represents a graph of the signal as a function of FIXconcentration, where the signal is generated by a FIX Padua-specificchromogenic activity assay.

FIG. 19 represents a schematic of strategies for the generation of FIXPadua specific Fabs and sequences of used panning and blocking peptides(L338 in FIX Padua is indicated in red and boxed, R338 in FIX wt isindicated in green and boxed)

FIG. 20 represents a schematic structure of purified mini antibodies(bivalent Fabs).

FIG. 21 represents (FIG. 21A) an X-ray structure of porcine FIXa anddemonstrates that Arg338 is located on the surface of heavy chain ofporcine FIXa and (FIG. 21B) a scheme of human FIX Padua and demonstratesLeu338.

FIG. 22 represents a graph demonstrating that purified Fabs bindspecifically to FIX Padua and not to FIX wt antigens. FIG. 22 representsa graph of an ELISA from purified bivalent Fabs against wt and Paduaantigens. Bivalent Fabs, obtained from different panning strategies weretested for specificity against wt and Padua peptide and protein. Resultsare represented as fold increase over background (FIG. 22A), and (FIG.22B) shows the scheme of the ELISA set up. The order of the columns isthe same as in the legend (i.e., Ab42 column is to the left). Fordetails, see the methods description in Example 5.

FIG. 23 represents a graph demonstrating that purified bivalent Fabsshow no cross reactivity to FIX wt in 20% human plasma matrix. FIG. 23represents a graph of an ELISA of purified bivalent Fabs in the presenceof 20% plasma. (FIG. 23A) Purified bivalent Fabs were coated andincubated with 20% human plasma containing 5 μg/mL FIX wt and thedenoted Padua FIX concentrations. Detection was performed with an HRPlabeled polyclonal goat anti FIX antibody (100 ng/ml). (FIG. 23B) Schemeof ELISA set up. The lines are in the same order as the legend.

FIG. 24 represents a graph demonstrating surface plasmon resonance (SPR)analysis of selected candidate Ab42. (FIG. 24A) His-tagged mini antibodyAb42 binds FIX Padua with a KD=59 nM (ka: 4.3×10⁴ 1/Ms; kd:0.0025311/s), (FIG. 24B) but not FIX wt on a NTA-BIAcore sensor chip. Dashedlines represent raw data, while solid lines indicated fitted data. Thelines are in the same order as the legend.

FIG. 25 represents an illustration of the immunoassay.

FIG. 26 represents a graph demonstrating assay selectivity asrepresented by concentration-response curves obtained for a purifiedFIXp sample and a fresh-frozen reference plasma preparation with anormal FIX concentration of 5 μg/mL demonstrate the selectivity of theassay. Human plasma showed essentially no response.

FIG. 27 represents a graph of the 6-point calibration curves, rangingfrom 0.85 to 27.1 ng FIXp/mL, had adequate linearity. Their accuracy wasdemonstrated by the correlation coefficients r, the low relative totalerrors (RTEs), and the results of the back-fitting approach.

FIG. 28 represents a set of graphs demonstrating the parallelism studyin normal and FIX-deficient plasma. The slopes of the dilution-responsecurves of the FIXp-spiked plasma differed by <5% from that obtained forthe buffer dilution series, indicating that the plasma matrix had noinfluence on the assay performance.

FIG. 29 represents a graph demonstrating the influence of Ca²⁺ on thesensitivity of the ELISA. A clear increase in sensitivity triggered byCa²⁺ is shown, very likely caused by the polyclonal detection antibodyand the Ca-dependent EGF domain of FIX.

FIG. 30 represents a set of graphs demonstrating the analysis ofcitrated plasma samples from a first patient treated with an AAV2/8viral vector in a phase 1/2 trial. The plasma sample obtained from thefirst patient was FIX cross-reactive material positive (CRM+).

FIG. 31 represents a set of graphs demonstrating the analysis ofcitrated plasma samples from a second patient treated with an AAV2/8viral vector in a phase 1/2 trial. The plasma sample obtained from thesecond patient was FIX cross-reactive material positive (CRM+).

FIG. 32 represents a set of graphs demonstrating the analysis ofcitrated plasma samples from a third patient treated with an AAV2/8viral vector in a phase 1/2 trial. The plasma sample obtained from thethird patient was not FIX cross-reactive material positive (CRM+).

DETAILED DESCRIPTION

Provided herein are binding constructs which specifically recognize FIXPadua with minimal or no cross-reactivity to wild-type FIX. In exemplaryaspects, the binding constructs bind to FIX Padua and do not bind towild-type (WT) Factor IX. In exemplary aspects, the binding constructsbind to FIX Padua (and not to WT Factor IX) in the presence of WT FactorIX. In exemplary aspects, the binding constructs bind to FIX Padua anddo not bind to one WT Factor IX and one or both of Factor II and FactorX, or any other mutated or modified forms thereof (except FIX Padua)under similar or the same conditions. In exemplary aspects, the bindingconstructs bind to FIX Padua and bind to neither Factor II nor Factor X.In exemplary aspects, the binding constructs bind to FIX Padua and bindto none of WT Factor IX, Factor II and Factor X. In exemplary aspects,the binding constructs bind to FIX Padua and (not to WT Factor IX,Factor II and Factor X) in the presence of WT Factor IX, Factor II andFactor X. In exemplary embodiments, the binding constructs bind anepitope of FIX Padua (SEQ ID NO: 1) even when present in a solutioncomprising levels of WT Factor IX, Factor II, and Factor X which arepresent in human plasma.

Epitopes

By “epitope” as used herein is meant the region of or within FIX Paduawhich is bound by the binding construct. In some embodiments, theepitope is a linear epitope. By “linear epitope” as used herein refersto the region of or within the FIX Padua which is bound by the bindingconstruct and which region is composed of contiguous amino acids of theamino acid sequence of the FIX Padua. The amino acids of a linearepitope are adjacent to each other in the primary structure of theFactor IX Padua. Accordingly, a linear epitope is a fragment or portionof the amino acid sequence of the antigen, i.e., FIX Padua.

In other exemplary embodiments, the epitope is a conformational orstructural epitope. By “conformational epitope” or “structural epitope”is meant an epitope which is composed of amino acids which are locatedin close proximity to one another when the Factor IX Padua is in itsproperly folded state. Unlike linear epitopes, the amino acids of aconformational or structural epitope need not be adjacent to each otherin the primary structure (i.e., amino acid sequence) of the FIX Padua. Aconformational or structural epitope is not necessarily made ofcontiguous amino acids of the amino acid sequence of the antigen (FIXp).

In exemplary embodiments, the binding construct binds to an epitope ofFIX Padua comprising the amino acid sequence of SEQ ID NO: 1, whereinthe epitope is a linear epitope within the amino acid sequence of SEQ IDNO: 1. In exemplary aspects, the linear epitope is within the amino acidsequence of DRATCLLSTKFT (SEQ ID NO: 3). In exemplary aspects, thelinear epitope comprises at least L-L of SEQ ID NO: 3. In exemplaryembodiments, the binding construct binds to the linear epitope of FIXPadua even in the present of WT Factor IX, Factor II, and/or Factor X.In exemplary embodiments, the binding construct binds to the epitope ofFIX Padua even in the presence of 5 μg/mL WT Factor IX. In exemplaryembodiments, the binding construct binds to the epitope of FIX Paduaeven in the presence of 5 μg/mL WT Factor IX and in a 20% human plasmamatrix.

In exemplary aspects, the binding construct does not bind to an epitopeof WT FIX. In exemplary aspects, the binding construct does not bind toan epitope within SEQ ID NO: 2 or within DRATCLRSTKFT (SEQ ID NO: 14) orwithin LVDRATCLRSTKFTIYNNMFCAGFH (SEQ ID NO: 15). In exemplary aspects,the binding construct does not bind to an epitope of WT FIX undersimilar or same conditions as when the binding construct binds to FIXPadua. In exemplary aspects, the binding construct does not bind to anepitope of WT FIX when in a solution comprising normal plasma levels ofWT FIX. In exemplary aspects, the binding construct does not bind to anepitope of WT FIX when in a solution (e.g., buffer) comprising 5 μg/mLWT FIX (e.g., a human plasma matrix comprising about 5 μg/mL WT FIX).

In exemplary embodiments, the binding construct binds to an epitope ofFIX Padua (SEQ ID NO: 1), wherein the epitope is a conformationalepitope of the folded structure of the amino acid sequenceLVDRATCLLSTKFTIYNNMFCAGFH (SEQ ID NO: 5). In exemplary embodiments, thebinding construct binds to an epitope of FIX Padua (SEQ ID NO: 1),wherein the epitope is a conformational epitope of the folded structureof the amino acid sequence LVDRATCLLSTKFTIYNNMFCAGFH (SEQ ID NO: 5),wherein the folded structure comprises a disulfide bridge. In exemplaryembodiments, the binding construct binds to the conformational epitopeof FIX Padua even in the present of WT Factor IX, Factor II, and/orFactor X. In exemplary embodiments, the binding construct binds to theepitope of FIX Padua even in the presence of 5 μg/mL WT Factor IX.

Affinity and Avidity

The binding constructs provided herein bind to FIX Padua in anon-covalent and reversible manner. In exemplary embodiments, thebinding strength of the binding construct to FIX Padua may be describedin terms of its affinity, a measure of the strength of interactionbetween the binding site of the binding construct and the epitope. Inexemplary aspects, the binding constructs provided herein havehigh-affinity for FIX Padua and thus will bind a greater amount of FIXPadua in a shorter period of time than low-affinity binding constructs.In exemplary aspects, the binding construct has an equilibriumassociation constant, KA, which is at least 10⁵ mol⁻¹, at least 10⁶mol⁻¹, at least 10⁷ mol⁻¹, at least 10⁸ mol⁻¹, at least 10⁹ mol⁻¹, or atleast 10¹⁰ mol⁻¹. In exemplary aspects, the binding constructs providedherein exhibit high affinity for FIX Padua in human plasma. In exemplaryaspects, the binding construct binds to the Factor IX Padua and does notbind to a WT Factor IX in a sample comprising human plasma. In exemplaryaspects, the binding construct binds to the Factor IX Padua and does notbind to a WT Factor IX in a sample comprising at least or about 5% humanplasma (e.g., a 5% human plasma matrix). In exemplary aspects, thebinding construct binds to the Factor IX Padua and does not bind to a WTFactor IX in a sample comprising at least or about 10% human plasma(e.g., a 10% human plasma matrix). In exemplary aspects, the bindingconstruct binds to the Factor IX Padua and does not bind to a WT FactorIX in a sample comprising at least or about 20% human plasma (e.g., a20% human plasma matrix). In exemplary aspects, the binding constructbinds to the Factor IX Padua and does not bind to a WT Factor IX in asample comprising at least or about 5% to about 40%, about 10% to about30%, or about 15% to about 20% human plasma. In exemplary aspects, thebinding construct binds to the Factor IX Padua even when a substantialamount of WT Factor IX is present in the sample. In exemplary aspects,the binding construct binds to the Factor IX Padua and does not bind toa WT Factor IX in a sample comprising an amount of human plasma (e.g.,at least or about 5% human plasma, at least or about 10% human plasma,at least or about 20% human plasma) and at least or about 1 μg/mL WTFactor IX or at least or about 2.5 μg/mL WT Factor IX or at least orabout 5 μg/m L WT Factor IX or at least or about 10 μg/m L WT Factor IX.In exemplary aspects, the binding construct binds to the Factor IX Paduaand does not bind to a WT Factor IX in a sample comprising othercoagulation factors, including, but not limited to Factor II, Factor V,Factor VI, Factor VII, Factor VIII, Factor X, Factor XI, Factor XII, andFactor XIII. In exemplary aspects, the binding construct binds to theFactor IX Padua and does not bind to a WT Factor IX and further does notbind to Factor II or Factor X. In exemplary aspects, the bindingconstruct binds to the Factor IX Padua and does not bind to a WT FactorIX and further binds to neither Factor II nor Factor X.

In exemplary embodiments, the binding strength of the binding constructto FIX Padua may be described in terms of its sensitivity. KD is theequilibrium dissociation constant, a ratio of k_(off)/k_(on), betweenthe binding construct and FIX Padua. KD and KA are inversely related.The KD value relates to the concentration of the binding construct (theamount of binding construct needed for a particular experiment) and sothe lower the KD value (lower concentration) the higher the affinity ofthe binding construct. In exemplary aspects, the binding strength of thebinding construct to FIX Padua may be described in terms of KD. Inexemplary aspects, the KD of the binding constructs provided herein forFIXp is about 1.0×10⁻⁶ or less, about 1.0×10⁻⁷ or less, about 1.0×10⁻⁸or less, about 1.0×10⁻⁹ or less, about 1.0×10⁻¹⁰ or less. In exemplaryaspects, the KD of the binding constructs provided herein is micromolar,nanomolar, picomolar or femtomolar. In exemplary aspects, the KD of thebinding constructs provided herein is within a range of about 10⁻⁴ to10⁻⁶ or 10⁻⁷ to 10⁻⁹ or 10⁻¹⁰ to 10⁻¹² or 10⁻¹³ to 10⁻¹⁶. In exemplaryaspects, the KD of the binding constructs provided herein is about 100nM or less. In certain aspects, the KD of the binding constructsprovided herein is about 20 nM to about 100 nM, about 25 nM to about 95nM, about 30 nM to about 90 nM, about 35 nM to about 85 nM, about 40 nMto about 80 nM, about 45 nM to about 75 nM, about 50 nM to about 70 nM,or about 55 nM to about 65 nM. In exemplary aspects, the KD of thebinding constructs is within a range of about 25 nM to about 75 nM. Inexemplary aspects, the KD of the binding constructs is within a range ofabout 50 nM to about 60 nM. In exemplary aspects, the KD of the bindingconstructs is about 56 nM.

Avidity gives a measure of the overall strength of an antibody-antigencomplex. It is dependent on three major parameters: affinity of thebinding construct for the epitope, valency of both the binding constructand FIX Padua, and structural arrangement of the parts that interact.The greater a binding construct's valency (number of antigen bindingsites), the greater the amount of antigen (FIX Padua) it can bind. Inexemplary aspects, the binding constructs have a strong avidity forFIXp. In exemplary aspects, the binding constructs are multivalent. Inexemplary aspects, the binding constructs are bivalent.

Structure

The binding constructs described herein may be engineered to have one ofa multitude of structures. In exemplary aspects, the binding constructsprovided herein have a structure of an antibody or antigen-bindingfragment thereof. In exemplary aspects, the binding constructs providedherein have a structure based on or derived from an antibody. Inexemplary aspects, the binding constructs provided herein have astructure of a synthetic antibody mimic, an engineered protein, or anaptamer, such as those described herein and in McEnaney et al.,“Chemically Synthesized Molecules with the Targeting and EffectorFunctions of Antibodies” J. Am. Chem. Soc., 136 (52): 18034-18043(2014); Binz and PlUckthun, “Engineered proteins as specific bindingreagents” Curr Opin Biotechnol. 16(4):459-69 (2005); and Roque et al.,“Antibodies and genetically engineered related molecules: production andpurification” Biotechnol Prog. 20(3):639-54 (2004).

Antibodies and Antigen-binding Fragments

In exemplary embodiments, the binding construct is an antibody. Theantibody may be any type of antibody, i.e., immunoglobulin, known in theart. In exemplary embodiments, the antibody is an antibody of class orisotype IgA, IgD, IgE, IgG, or IgM. In exemplary embodiments, theantibody described herein comprises one or more alpha, delta, epsilon,gamma, and/or mu heavy chains. In exemplary embodiments, the antibodydescribed herein comprises one or more kappa or light chains. Inexemplary aspects, the antibody is an IgG antibody and optionally is oneof the four human subclasses: IgG1, IgG2, IgG3 and IgG4.

Also, the antibody in some embodiments is a monoclonal antibody. Inother embodiments, the antibody is a polyclonal antibody.

In some embodiments, the antibody is structurally similar to or derivedfrom a naturally-occurring antibody, e.g., an antibody isolated and/orpurified from a mammal, e.g., mouse, rabbit, goat, horse, chicken,hamster, human, and the like. In this regard, the antibody may beconsidered as a mammalian antibody, e.g., a mouse antibody, rabbitantibody, goat antibody, horse antibody, chicken antibody, hamsterantibody, human antibody, and the like. In exemplary aspects, theantibody comprises sequence of only mammalian antibodies. Methods ofproducing such antibodies are known in the art, some of which aredescribed further herein under the section entitled “Methods of AntibodyProduction.” In exemplary aspects, the binding construct is a fullyhuman antibody, or does not comprise sequences of non-human antibodies.

In some embodiments, the antibody is a genetically-engineered antibodyand does not occur in nature. In exemplary embodiments, the antibody isa single chain antibody, a humanized antibody, a chimeric antibody, aCDR-grafted antibody, a humaneered antibody, a bispecific antibody, atrispecific antibody, and the like. Genetic engineering techniques alsoprovide the ability to make fully human antibodies from a non-humansource.

In some aspects, the genetically-engineered antibody is a single chainantibody (SCA) specific for FIX Padua. Methods of making SCAs are knownin the art. See, for example, Davis et al., Nature Biotechnology 9:165-169 (1991).

In some aspects, the antibody is a chimeric antibody. The term “chimericantibody” is used herein to refer to an antibody containing constantdomains from one species and the variable domains from a second, or moregenerally, containing stretches of amino acid sequence from at least twospecies. In particular aspects, the chimeric antibody binds to FIXPadua.

In some aspects, the antibody is a humanized antibody. The term“humanized” when used in relation to antibodies refers to antibodieshaving at least CDR regions from a non-human source which are engineeredto have a structure and immunological function more similar to truehuman antibodies than the original source antibodies. For example,humanizing can involve grafting CDR from a non-human antibody, such as amouse antibody, into a human antibody. Humanizing also can involveselect amino acid substitutions to make a non-human sequence look morelike a human sequence.

Use of the terms “chimeric or humanized” herein is not meant to bemutually exclusive, and rather, is meant to encompass chimericantibodies, humanized antibodies, and chimeric antibodies that have beenfurther humanized. Except where context otherwise indicates, statementsabout (properties of, uses of, testing of, and so on) chimericantibodies apply to humanized antibodies, and statements about humanizedantibodies pertain also to chimeric antibodies. Likewise, except wherecontext dictates, such statements also should be understood to beapplicable to antibodies and antigen-binding fragments of suchantibodies.

In some aspects, the antibody is a Humaneered™ antibody. Humaneeringtechnology is a proprietary method of KaloBios Pharmaceuticals, Inc.(South San Francisco, Calif.) for converting non-human antibodies intoengineered human antibodies. Humaneered™ antibodies have high affinity,and highly similar to human germ line antibody sequences. See, e.g.,Tomasevic et al., Growth Factors 32: 223-235 (2014).

In some aspects, the antibody is a CDR-grafted antibody specific for FIXPadua. Methods of making CDR-grafted antibodies are known in the art.See, for example, Lo, Benny, Antibody Engineering: Methods andProtocols, Volume 248 (2004), which is incorporated by reference in itsentirety.

In exemplary embodiments, the antibody is engineered to be bispecific,trispecific, or multi-specific, and the antibody comprises two or moredistinct antigen-binding regions. In some aspects, the antibody is abispecific or trispecific antibody specific for FIX Padua. Methods ofmaking bispecific or trispecific antibodies are known in the art. See,for example, Marvin and Zhu, Acta Pharmacologica Sinica 26: 649-658(2005) and U.S. Pat. No. 6,551,592. In exemplary aspects, the bindingconstruct is a bi-specific antigen-binding construct specific for afirst epitope of FIX Padua and a second epitope of FIX Padua. Inexemplary embodiments, the antibody is quadroma, heterodimericbispecific antibody, bispecific antibody fusion, bispecific antibodyfragment, a bispecific T-cell engager (BiTE), or a multi-specificantibody. In exemplary embodiments, the antibody is engineered to bebivalent, trivalent, or multivalent. See, e.g., Cuesta et al.,“Multivalent antibodies: when design surpasses evolution” Trends inBiotechnology 28, 355-362 (2010); Holliger et al., “Engineered antibodyfragments and the rise of single domains” Nat. Biotechnol. 23, 1126-1136(2005); Chan et al., “Therapeutic antibodies for autoimmunity andinflammation” Nat Rev Immunol 10, 301-316 (2010); Byrne et al., “A taleof two specificities: bispecific antibodies for therapeutic anddiagnostic applications” Trends Biotechnol. 31, 621-632 (2013). In someembodiments, the antibody is in monomeric form, while in otherembodiments, the antibody is conjugated to one or more antibodies (e.g.,each of which recognize the same epitope of the first antibody).Accordingly, in some aspects, the antibody is in dimeric, polymeric,oligomeric, or multimeric form.

In exemplary aspects, the binding construct is an antigen-bindingfragment of an antibody or comprises an antigen-binding fragment of anantibody. The antigen-binding fragment (also referred to herein as“antigen-binding portion”) may be an antigen-binding fragment of any ofthe antibodies described herein. The antigen-binding fragment can be anypart of an antibody that has at least one antigen binding site,including, but not limited to, Fab, F(ab′)2, a monospecific orbispecific Fab2, a trispecific Fabs, a monovalent IgG, scFv, dsFv,scFv-Fc, bispecific diabodies, trispecific triabodies, minibodies, or afragment of IgNAR (e.g., V-NAR), or a fragment of hclgG (e.g., VhH), orbis-scFvs, fragments expressed by a Fab expression library, and thelike. In exemplary aspects, the antigen-binding fragment is a domainantibody, VhH domain, V-NAR domain, VH domain, VL domain, or the like.Antibody fragments of the disclosure, however, are not limited to theseexemplary types of antibody fragments. In exemplary aspects, the bindingconstruct comprises a Fab fragment. In exemplary aspects, the bindingconstruct comprises two Fab fragments. In exemplary aspects, the bindingconstruct comprises two Fab fragments connected via a linker. Inexemplary aspects, the binding construct comprises or is a minibodycomprising two Fab fragments. In exemplary aspects, the bindingconstruct comprises or is a minibody comprising two Fab fragments joinedvia a linker. Minibodies are known in the art. See, e.g., Hu et al.,Cancer Res 56: 3055-3061 (1996). In exemplary aspects, the bindingconstruct comprises or is a minibody comprising two Fab fragments joinedvia a linker, optionally, comprising an alkaline phosphatase domain.

A domain antibody comprises a functional binding unit of an antibody,and can correspond to the variable regions of either the heavy (VH) orlight (VL) chains of antibodies. A domain antibody can have a molecularweight of approximately 13 kDa, or approximately one-tenth of a fullantibody. Domain antibodies may be derived from full antibodies such asthose described herein.

The binding constructs in some embodiments are monomeric or polymeric,bispecific or trispecific, bivalent or trivalent. In exemplary aspects,the binding construct provided herein is monospecific. In exemplaryaspects, the binding construct provided herein is bispecific. Inexemplary aspects, the binding construct provided herein is fully human.In exemplary aspects, the binding construct comprises two Fab fragmentsand is bivalent. In exemplary aspects, the binding construct is ahomodimer of two Fab fragments that are identical in structure. Thus, inexemplary aspects, the binding construct is bivalent but monospecificfor FIX Padua. In exemplary aspects, the homodimer is dimerized via ahelix-turn-helix motif. In exemplary aspects, the binding construct is ahomodimer of two Fab mini antibodies that are identical in structure.Thus, in exemplary aspects, the binding construct is bivalent butmonospecific for FIX Padua. In exemplary aspects, the homodimer of twoFab mini antibodies are dimerized via an alkaline phosphatase domain.

Antibody fragments that contain the antigen-binding, or idiotype, of theantibody molecule may be generated by techniques known in the art. Forexample, such fragments include, but are not limited to, the F(ab′)₂fragment which may be produced by pepsin digestion of the antibodymolecule; the Fab′ fragments which may be generated by reducing thedisulfide bridges of the F(ab′)₂ fragment; and the two Fab′ fragmentswhich may be generated by treating the antibody molecule with papain anda reducing agent.

A single-chain variable region fragment (sFv) antibody fragment, whichconsists of a truncated Fab fragment comprising the variable (V) domainof an antibody heavy chain linked to a V domain of a light antibodychain via a synthetic peptide, can be generated using routinerecombinant DNA technology techniques (see, e.g., Janeway et al.,supra). Similarly, disulfide-stabilized variable region fragments (dsFv)can be prepared by recombinant DNA technology (see, e.g., Reiter et al.,Protein Engineering, 7, 697-704 (1994)).

Recombinant antibody fragments, e.g., scFvs, can also be engineered toassemble into stable multimeric oligomers of high binding avidity andspecificity to different target antigens. Such diabodies (dimers),triabodies (trimers) or tetrabodies (tetramers) are well known in theart, see e.g., Kortt et al., Biomol Eng. 2001 18:95-108, (2001) andTodorovska et al., J Immunol Methods. 248:47-66, (2001).

Bispecific antibodies (bscAb) are molecules comprising two single-chainFv fragments joined via a glycine-serine linker using recombinantmethods. The V light-chain (VL) and V heavy-chain (VH) domains of twoantibodies of interest in exemplary embodiments are isolated usingstandard PCR methods. The VL and VH cDNA's obtained from each hybridomaare then joined to form a single-chain fragment in a two-step fusionPCR. Bispecific fusion proteins are prepared in a similar manner.Bispecific single-chain antibodies and bispecific fusion proteins areantibody substances included within the scope of the present disclosure.Exemplary bispecific antibodies are taught in U.S. Patent ApplicationPublication No. 2005-0282233A1 and International Patent ApplicationPublication No. WO 2005/087812, both applications of which areincorporated herein by reference in their entirety.

In exemplary embodiments, the binding construct is a biparatopicantibody, or a biparatopic antigen-binding fragment thereof, having thecapability of binding two different non-overlapping epitopes on the sametarget antigen molecule. By simultaneously binding to the same cellsurface targets, biparatopic antibodies and biparatopic antigen-bindingfragments thereof may result in enhanced binding avidity, leading topreferential (strong) binding to only cells that express the targets,thus fine-tuning the antibody selectivity. It has been demonstrated thatbiparatopic antibodies or biparatopic antigen-binding fragments thereof,by simultaneously binding to two different epitopes on the same targetmolecule, could even potentially acquire new functionality that couldnot be achieved with the parent antibodies (or antigen-bindingfragments) when used alone or in combination. In exemplary aspects, thebinding constructs provided herein are biparatopic for FIX Padua.

In exemplary embodiments, the antigen-binding fragment is engineered tobe bispecific, trispecific, or multi-specific. In exemplary aspects, theantigen-binding fragment comprises two or more distinct antigen-bindingregions. In some aspects, the antigen-binding fragment is a bispecificor trispecific antibody specific for FIX Padua and at least one otherantigen. In exemplary aspects, the binding construct is a bi-specificantigen-binding fragment specific for a first epitope of FIX Padua and asecond epitope of FIX Padua. In exemplary embodiments, theantigen-binding fragment is engineered to be bivalent, trivalent, ormultivalent. In exemplary embodiments, the binding construct is abivalent Fab fragment monospecific for FIX Padua. In some embodiments,the antigen-binding fragment is in monomeric form, while in otherembodiments, the antigen-binding fragment is conjugated to one or moreantigen-binding fragments (e.g., each of which recognize the sameepitope of the first antigen-binding fragment). Accordingly, in someaspects, the antigen-binding fragment is dimerized, polymerized,oligomerized, or multimerized. In exemplary aspects, the bindingconstruct is a dimerized Fab fragment. In exemplary aspects, the bindingconstruct is a fully human dimerized Fab fragment. In exemplary aspects,the binding construct is dimerized via a helix-turn-helix motif. Inexemplary embodiments, the antigen-binding fragment is engineered to bebivalent, trivalent, or multivalent. In exemplary embodiments, thebinding construct is a dimerized bivalent Fab fragment monospecific forFIX Padua, wherein the binding construct is dimerized via ahelix-turn-helix motif.

In exemplary aspects, the binding construct, e.g., antibody orantigen-binding fragment thereof, comprises the amino acid sequences of:SSYAIS (SEQ ID NO: 6); GIVPAFGTANYAQKFQG (SEQ ID NO: 7); SWGVISFAY (SEQID NO: 8); RASQDISSYLN (SEQ ID NO: 9); AASNLQS (SEQ ID NO: 10); andMQYDSLPFTF (SEQ ID NO: 11). In exemplary aspects, one or more aminoacids are present between each of SEQ ID NOs: 6-11. In exemplaryaspects, the binding construct, e.g., antibody or antigen-bindingfragment thereof, comprises the sequence of SEQ ID NO: 24 or SEQ ID NO:25 or both SEQ ID NOs: 24 and 25. In exemplary aspects, the bindingconstruct, e.g., antibody or antigen-binding fragment thereof, comprisesthe sequence of SEQ ID NO: 26 or SEQ ID NO: 27 or both SEQ ID NOs: 26and 27. In exemplary aspects, the amino acid sequence of the bindingconstruct, e.g., antibody or antigen-binding fragment thereof, comprisesadditional sequences of, e.g., linker(s), expression tags (e.g., Histags, FLAG tags, myc tags, fluorescent proteins (e.g., green fluorescentprotein, blue fluorescent protein, red fluorescent protein, yellowfluorescent protein, cyan fluorescent protein, enhanced greenfluorescent protein, and the like). In exemplary aspects, the bindingconstruct, e.g., antibody or antigen-binding fragment thereof, comprisesthe sequence of a His tag and/or a FLAG tag. In exemplary aspects, theFLAG tag comprises a sequence of SEQ ID NO: 12. In exemplary aspects,the His tag comprises a sequence of SEQ ID NO: 13. In exemplary aspects,the binding construct, e.g., antibody or antigen-binding fragmentthereof, comprises a linker. In exemplary aspects, the bindingconstruct, e.g., antibody or antigen-binding fragment thereof, comprisesa synthetic double helix loop helix motif, such as that described inHaylock et al., Int J. Oncol. 48(2): 461-470 (2016) or Wang et al.,Anal. Chem. 78: 997-1004 (2006). In exemplary aspects, the bindingconstruct, e.g., antibody or antigen-binding fragment thereof, comprisesa constant antibody domain. Such antibody domains are described in Hu etal., Cancer Res 56: 3055-3061 (1996) and McGregor et al., Mol Immuno 31:219-226 (1994). In exemplary aspects, the binding construct, e.g.,antibody or antigen-binding fragment thereof, comprises a bacterialalkaline phosphatase domain, such as that described in Wang et al.(2006), supra. In exemplary aspects, the binding construct, e.g.,antibody or antigen-binding fragment thereof, comprises the sequence ofSEQ ID NO: 28 or SEQ ID NO: 27 or both SEQ ID NOs: 28 and 27.

Aptamers

In some embodiments, the binding construct is an analog of an antibody.In some aspects, the binding construct is an aptamer. Recent advances inthe field of combinatorial sciences have identified short polymersequences (e.g., oligonucleic acid or peptide molecules) with highaffinity and specificity to a given target. For example, SELEXtechnology has been used to identify DNA and RNA aptamers with bindingproperties that rival mammalian antibodies, the field of immunology hasgenerated and isolated antibodies or antibody fragments which bind to amyriad of compounds and phage display has been utilized to discover newpeptide sequences with very favorable binding properties. Based on thesuccess of these molecular evolution techniques, it is certain thatmolecules can be created which bind to any target molecule. A loopstructure is often involved with providing the desired bindingattributes, as in the case of aptamers which often utilize hairpin loopscreated from short regions without complimentary base pairing, andnaturally derived antibodies that utilize combinatorial arrangement oflooped hyper-variable regions and new phage display libraries utilizingcyclic peptides that have shown improved results when compared to linearpeptide phage display results. For more on aptamers, see, generally,Gold, L., Singer, B., He, Y. Y., Brody. E., “Aptamers As Therapeutic AndDiagnostic Agents,” J. Biotechnol. 74:5-13 (2000). Relevant techniquesfor generating aptamers may be found in U.S. Pat. No. 6,699,843, whichis incorporated by reference in its entirety.

Methods of Antibody or Antigen-binding Fragment Production

Suitable methods of making antibodies are known in the art. Forinstance, standard hybridoma methods are described in, e.g., Harlow andLane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and CA.Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, NewYork, N.Y. (2001)).

Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogen comprising a polypeptide of the present disclosure andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. In some aspects, ananimal used for production of anti-antisera is a non-human animalincluding rabbits, mice, rats, hamsters, goat, sheep, pigs or horses.Because of the relatively large blood volume of rabbits, a rabbit is apreferred choice for production of polyclonal antibodies. In anexemplary method for generating a polyclonal antisera immunoreactivewith the chosen FIX Padua epitope, 50 μg of FIX Padua antigen isemulsified in Freund's Complete Adjuvant for immunization of rabbits. Atintervals of, for example, 21 days, 50 μg of epitope are emulsified inFreund's Incomplete Adjuvant for boosts. Polyclonal antisera may beobtained, after allowing time for antibody generation, simply bybleeding the animal and preparing serum samples from the whole blood.

Monoclonal antibodies for use in the methods of the disclosure may beprepared using any technique which provides for the production ofantibody molecules by continuous cell lines in culture. These includebut are not limited to the hybridoma technique originally described byKoehler and Milstein (Nature 256: 495-497, 1975), the human B-cellhybridoma technique (Kosbor et al., Immunol Today 4:72, 1983; Cote etal., Proc Natl Acad Sci 80: 2026-2030, 1983) and the EBV-hybridomatechnique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan RLiss Inc, New York N.Y., pp 77-96, (1985).

Briefly, in exemplary embodiments, to generate monoclonal antibodies, amouse is injected periodically with recombinant FIX Padua against whichthe antibody is to be raised (e.g., 10-20 μg emulsified in Freund'sComplete Adjuvant). The mouse is given a final pre-fusion boost of FIXPadua in PBS, and four days later the mouse is sacrificed and its spleenremoved. The spleen is placed in 10 ml serum-free RPMI 1640, and asingle cell suspension is formed by grinding the spleen between thefrosted ends of two glass microscope slides submerged in serum-free RPMI1640, supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 100units/ml penicillin, and 100 μg/ml streptomycin (RPMI) (Gibco, Canada).The cell suspension is filtered through sterile 70-mesh Nitex cellstrainer (Becton Dickinson, Parsippany, N.J.), and is washed twice bycentrifuging at 200 g for 5 minutes and resuspending the pellet in 20 mlserum-free RPMI. Splenocytes taken from three naïve Balb/c mice areprepared in a similar manner and used as a control. NS-1 myeloma cells,kept in log phase in RPMI with 11% fetal bovine serum (FBS) (HycloneLaboratories, Inc., Logan, Utah) for three days prior to fusion, arecentrifuged at 200 g for 5 minutes, and the pellet is washed twice.

Spleen cells (1×10⁸) are combined with 2.0×10⁷ NS-1 cells andcentrifuged, and the supernatant is aspirated. The cell pellet isdislodged by tapping the tube, and 1 ml of 37° C. PEG 1500 (50% in 75 mMHepes, pH 8.0) (Boehringer Mannheim) is added with stirring over thecourse of 1 minute, followed by the addition of 7 ml of serum-free RPMIover 7 minutes. An additional 8 ml RPMI is added and the cells arecentrifuged at 200 g for 10 minutes. After discarding the supernatant,the pellet is resuspended in 200 ml RPMI containing 15% FBS, 100 μMsodium hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine (HAT) (Gibco),25 units/ml IL-6 (Boehringer Mannheim) and 1.5×10⁶ splenocytes/ml andplated into 10 Corning flat-bottom 96-well tissue culture plates(Corning, Corning N.Y.).

On days 2, 4, and 6, after the fusion, 100 μl of medium is removed fromthe wells of the fusion plates and replaced with fresh medium. On day 8,the fusion is screened by ELISA, testing for the presence of mouse IgGbinding to FIX Padua as follows. Immulon 4 plates (Dynatech, Cambridge,Mass.) are coated for 2 hours at 37° C. with 100 ng/well of EGFR dilutedin 25 mM Tris, pH 7.5. The coating solution is aspirated and 200 μl/wellof blocking solution (0.5% fish skin gelatin (Sigma) diluted in CMF-PBS)is added and incubated for 30 min. at 37° C. Plates are washed threetimes with PBS with 0.05% Tween 20 (PBST) and 50 μl culture supernatantis added. After incubation at 37° C. for 30 minutes, and washing asabove, 50 μl of horseradish peroxidase conjugated goat anti-mouseIgG(fc) (Jackson ImmunoResearch, West Grove, Pa.) diluted 1:3500 in PBSTis added. Plates are incubated as above, washed four times with PBST,and 100 μl substrate, consisting of 1 mg/ml o-phenylene diamine (Sigma)and 0.1 μl/ml 30% H₂O₂ in 100 mM Citrate, pH 4.5, are added. The colorreaction is stopped after 5 minutes with the addition of 50 μl of 15%H₂SO₄. A₄₉₀ is read on a plate reader (Dynatech).

Selected fusion wells are cloned twice by dilution into 96-well platesand visual scoring of the number of colonies/well after 5 days. Themonoclonal antibodies produced by hybridomas are isotyped using theIsostrip system (Boehringer Mannheim, Indianapolis, Ind.).

When the hybridoma technique is employed, myeloma cell lines may beused. Such cell lines suited for use in hybridoma-producing fusionprocedures preferably are non-antibody-producing, have high fusionefficiency, and enzyme deficiencies that render them incapable ofgrowing in certain selective media which support the growth of only thedesired fused cells (hybridomas). For example, where the immunizedanimal is a mouse, one may use P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 4 1,Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and 5194/15XX0 Bul; forrats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 46210; and U-266,GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection withcell fusions.

Depending on the host species, various adjuvants may be used to increaseimmunological response. Such adjuvants include but are not limited toFreund's, mineral gels such as aluminum hydroxide, and surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG(bacilli Calmette-Guerin) and Corynebacterium parvum are potentiallyuseful human adjuvants.

Alternatively, other methods, such as EBV-hybridoma methods (Haskard andArcher, J. Immunol. Methods, 74(2), 361-67 (1984), and Roder et al.,Methods Enzymol., 121, 140-67 (1986)), and bacteriophage vectorexpression systems (see, e.g., Huse et al., Science, 246, 1275-81(1989)) are known in the art. Further, methods of producing antibodiesin non-human animals are described in, e.g., U.S. Pat. Nos. 5,545,806,5,569,825, and 5,714,352, and U.S. Patent Application Publication No.2002/0197266 A1).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inOrlandi et al (Proc Natl Acad Sci 86: 3833-3837; 1989), and Winter G andMilstein C (Nature 349: 293-299, 1991). If the full sequence of theantibody or antigen-binding fragment is known, then methods of producingrecombinant proteins may be employed. See, e.g., “Protein production andpurification” Nat Methods 5(2): 135-146 (2008).

Phage display also can be used to generate the antibody of the presentdisclosures. In this regard, phage libraries encoding antigen-bindingvariable (V) domains of antibodies can be generated using standardmolecular biology and recombinant DNA techniques (see, e.g., Sambrook etal. (eds.), Molecular Cloning, A Laboratory Manual, 3rd Edition, ColdSpring Harbor Laboratory Press, New York (2001)). Phage encoding avariable region with the desired specificity are selected for specificbinding to the desired antigen, and a complete or partial antibody isreconstituted comprising the selected variable domain. Nucleic acidsequences encoding the reconstituted antibody are introduced into asuitable cell line, such as a myeloma cell used for hybridomaproduction, such that antibodies having the characteristics ofmonoclonal antibodies are secreted by the cell (see, e.g., Janeway etal., supra, Huse et al., supra, and U.S. Pat. No. 6,265,150). Relatedmethods also are described in U.S. Pat. Nos. 5,403,484; 5,571,698;5,837,500; 5,702,892. The techniques described in U.S. Pat. Nos.5,780,279; 5,821,047; 5,824,520; 5,855,885; 5,858,657; 5,871,907;5,969,108; 6,057,098; and 6,225,447.

Antibodies can be produced by transgenic mice that are transgenic forspecific heavy and light chain immunoglobulin genes. Such methods areknown in the art and described in, for example U.S. Pat. Nos. 5,545,806and 5,569,825, and Janeway et al., supra.

Methods for generating humanized antibodies are well known in the artand are described in detail in, for example, Janeway et al., supra, U.S.Pat. Nos. 5,225,539, 5,585,089 and 5,693,761, European Patent No.0239400 BI, and United Kingdom Patent No. 2188638. Humanized antibodiescan also be generated using the antibody resurfacing technologydescribed in U.S. Pat. No. 5,639,641 and Pedersen et al., J. Mol. Biol,235, 959-973 (1994). A preferred chimeric or humanized antibody has ahuman constant region, while the variable region, or at least a CDR, ofthe antibody is derived from a non-human species. Methods for humanizingnon-human antibodies are well known in the art. (See U.S. Pat. Nos.5,585,089, and 5,693,762.) Generally, a humanized antibody has one ormore amino acid residues introduced into its framework region from asource which is non-human. Humanization can be performed, for example,using methods described in Jones et al. (Nature 321: 522-525, 1986),Riechmann et al., (Nature, 332: 323-327, 1988) and Verhoeyen et al.(Science 239:1534-1536, 1988), by substituting at least a portion of arodent complementarity-determining region (CDRs) for the correspondingregions of a human antibody. Numerous techniques for preparingengineered antibodies are described, e.g., in Owens and Young, J.Immunol. Meth., 168:149-165 (1994). Further changes can then beintroduced into the antibody framework to modulate affinity orimmunogenicity.

Techniques developed for the production of “chimeric antibodies”, thesplicing of mouse antibody genes to human antibody genes to obtain amolecule with appropriate antigen specificity and biological activity,can be used (Morrison et al., Proc Natl Acad Sci 81: 6851-6855 (1984);Neuberger et al., Nature 312: 604-608 (1984); Takeda et al., Nature 314:452-454 (1985)). Alternatively, techniques described for the productionof single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted toproduce EGFR- or HSP90-specific single chain antibodies.

Likewise, using techniques known in the art to isolate CDRs,compositions comprising CDRs are generated. Complementarity determiningregions are characterized by six polypeptide loops, three loops for eachof the heavy or light chain variable regions. The amino acid position ina CDR is defined by Kabat et al., “Sequences of Proteins ofImmunological Interest,” U.S. Department of Health and Human Services,(1983), which is incorporated herein by reference. For example,hypervariable regions of human antibodies are roughly defined to befound at residues 28 to 35, from 49-59 and from residues 92-103 of theheavy and light chain variable regions (Janeway and Travers,Immunobiology, 2^(nd) Edition, Garland Publishing, New York, (1996)).The murine CDRs also are found at approximately these amino acidresidues. It is understood in the art that CDR regions may be foundwithin several amino acids of these approximated residues set forthabove. An immunoglobulin variable region also consists of four“framework” regions surrounding the CDRs (FR1-4). The sequences of theframework regions of different light or heavy chains are highlyconserved within a species, and are also conserved between human andmurine sequences.

Compositions comprising one, two, and/or three CDRs of a heavy chainvariable region or a light chain variable region of a monoclonalantibody are generated. Techniques for cloning and expressing nucleotideand polypeptide sequences are well-established in the art (see e.g.Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) Edition,Cold Spring Harbor, N.Y. (1989)). The amplified CDR sequences areligated into an appropriate expression vector. The vector comprisingone, two, three, four, five and/or six cloned CDRs optionally containsadditional polypeptide encoding regions linked to the CDR.

Framework regions (FR) of a murine antibody are humanized bysubstituting compatible human framework regions chosen from a largedatabase of human antibody variable sequences, including over twelvehundred human VH sequences and over one thousand VL sequences. Thedatabase of antibody sequences used for comparison is downloaded fromAndrew C. R. Martin's KabatMan web page(http://www.rubic.rdg.ac.uk/abs/). The Kabat method for identifying CDRprovides a means for delineating the approximate CDR and frameworkregions from any human antibody and comparing the sequence of a murineantibody for similarity to determine the CDRs and FRs. Best matchedhuman VH and VL sequences are chosen on the basis of high overallframework matching, similar CDR length, and minimal mismatching ofcanonical and VH/VL contact residues. Human framework regions mostsimilar to the murine sequence are inserted between the murine CDR.Alternatively, the murine framework region may be modified by makingamino acid substitutions of all or part of the native framework regionthat more closely resemble a framework region of a human antibody.

Additionally, another useful technique for generating antibodies for usein the present disclosure may be one which uses a rational design typeapproach. The goal of rational design is to produce structural analogsof biologically active polypeptides or compounds with which theyinteract (agonists, antagonists, inhibitors, peptidomimetics, bindingpartners, etc.). In one approach, one would generate a three-dimensionalstructure for the antibodies or an epitope binding fragment thereof.This could be accomplished by x-ray crystallography, computer modelingor by a combination of both approaches. An alternative approach,“alanine scan,” involves the random replacement of residues throughoutmolecule with alanine, and the resulting effect on function determined.

Chemically constructed bispecific antibodies may be prepared bychemically cross-linking heterologous Fab or F(ab′)₂ fragments by meansof chemicals such as heterobifunctional reagentsuccinimidyl-3-(2-pyridyldithiol)-propionate (SPDP, Pierce Chemicals,Rockford, Ill.). The Fab and F(ab′)₂ fragments can be obtained fromintact antibody by digesting it with papain or pepsin, respectively(Karpovsky et al., J. Exp. Med. 160:1686-701 (1984); Titus et al., J.Immunol., 138:4018-22 (1987)).

Methods of testing antibodies for the ability to bind to the epitope ofthe FIX Padua regardless of how the antibodies are produced are known inthe art and include any antibody-antigen binding assay, such as, forexample, radioimmunoassay (RIA), ELISA, Western blot,immunoprecipitation, surface plasmon resonance, and competitiveinhibition assays (see, e.g., Janeway et al., infra, and U.S. PatentApplication Publication No. 2002/0197266).

Polypeptides

A polypeptide comprising an amino acid sequence comprising each of SEQID NOs: 6-11 is further provided herein. The polypeptide binds to FIXpand not to WT FIX, e.g., the polypeptide binds to FIXp only even in thepresence of WT FIX, optionally, even in the presence of othercoagulation factors, e.g., Factor II and Factor X. In exemplary aspects,the polypeptide binds to FIXp in a sample comprising at least 5%, atleast 10% or at least 20% human plasma. In exemplary aspects, one ormore amino acids are present between each of SEQ ID NOs: 6-11. Inexemplary aspects, the polypeptide comprises the sequence of SEQ ID NO:24 or SEQ ID NO: 25 or both SEQ ID NOs: 24 and 25. In exemplary aspects,the polypeptide comprises the sequence of SEQ ID NO: 26 or SEQ ID NO: 27or both SEQ ID NOs: 26 and 27. In exemplary aspects, the amino acidsequence of the polypeptide comprises additional sequences of, e.g.,linker(s), expression tags (e.g., His tags, FLAG tags, myc tags,fluorescent proteins (e.g., green fluorescent protein, blue fluorescentprotein, red fluorescent protein, yellow fluorescent protein, cyanfluorescent protein, enhanced green fluorescent protein, and the like).In exemplary aspects, the polypeptide comprises the sequence of a Histag and/or a FLAG tag. In exemplary aspects, the FLAG tag comprises asequence of SEQ ID NO: 12. In exemplary aspects, the His tag comprises asequence of SEQ ID NO: 13. In exemplary aspects, the polypeptidecomprises a linker. In exemplary aspects, the polypeptide comprises asequence of a synthetic double helix loop helix motif, such as thatdescribed in Haylock et al., Int J. Oncol. 48(2): 461-470 (2016) or Wanget al., Anal. Chem. 78: 997-1004 (2006). In exemplary aspects, thepolypeptide comprises a sequence of a constant antibody domain. Suchantibody domains are described in Hu et al., Cancer Res 56: 3055-3061(1996) and McGregor et al., Mol Immuno 31: 219-226 (1994). In exemplaryaspects, the polypeptide comprises a bacterial alkaline phosphatasedomain, such as that described in Wang et al. (2006), supra. Inexemplary aspects, the polypeptide comprises the sequence of SEQ ID NO:28 or SEQ ID NO: 27 or both SEQ ID NOs: 28 and 27.

Conjugates

The binding constructs described herein can be modified, for instance,by glycosylation, amidation, carboxylation, or phosphorylation, or bythe creation of acid addition salts, amides, esters, in particularC-terminal esters, and N-acyl derivatives. The binding construct alsocan be modified to create derivatives by forming covalent or noncovalentcomplexes with other moieties, i.e., conjugates. Covalently boundcomplexes can be prepared by linking the chemical moieties to functionalgroups on the side chains of amino acids comprising the bindingconstruct, or at the N- or C-terminus.

In some embodiments, the binding constructs of the present disclosureare attached, linked, joined, or conjugated to a second moiety (e.g., aheterologous moiety) and the resulting product is a conjugate.Accordingly, provided herein are conjugates comprising the bindingconstructs described herein (covalently or non-covalently) linked to aheterologous moiety. As used herein, the term “heterologous moiety”refers to any molecule (chemical or biochemical, naturally-occurring ornon-coded) which is different from the binding constructs of theinvention. Exemplary heterologous moieties include, but are not limitedto, a polymer, a carbohydrate, a lipid, a nucleic acid, anoligonucleotide, a DNA or RNA, an amino acid, peptide, polypeptide,protein, therapeutic agent, (e.g., a cytotoxic agent, cytokine), adiagnostic agent or a detecting agent.

In some embodiments, the binding constructs are chemically modified withvarious heterologous moieties. In some embodiments, the chemicalmodifications impart additional desirable characteristics as discussedherein. Chemical modifications in some aspects take a number ofdifferent forms such as heterologous peptides, polysaccharides, lipids,radioisotopes, non-standard amino acid resides and nucleic acids, metalchelates, and various cytotoxic agents.

In some embodiments, the binding constructs are fused to heterologouspeptides to confer various properties, e.g., increased solubility and/orstability and/or half-life, resistance to proteolytic cleavage,modulation of clearance, targeting to particular cell or tissue types.In some embodiments, the binding constructs are linked to an Fc domainof IgG or other immunoglobulin. In some embodiments, the bindingconstruct is fused to alkaline phosphatase (AP). Methods for making Fcor AP fusion constructs are found in International Patent PublicationNo. WO 02/060950. By fusing the binding construct with protein domainsthat have specific properties (e.g., half-life, bioavailability) it ispossible to confer these properties to the binding construct of theinvention.

The binding constructs can be conjugated to a detecting agent (e.g., adetectable label or a reporter group), including, but not limited to aradiolabel, a fluorescent label, an enzyme (e.g., that catalyzes acalorimetric or fluorometric reaction), a substrate, a solid matrix, ora carrier (e.g., biotin or avidin). In exemplary aspects, thefluorescent label comprises a rhodamine dye, fluorescein dye and/or acyanine dye. In exemplary embodiments, the fluorescent label comprises aset of dyes, e.g., a rhodamine dye, TAMRA, and a fluorescein dye, FAM.In another embodiment, the fluorescent label comprises of a set offluorescent dyes formed by selecting two or more dyes from the groupconsisting of Oregon Green 488, Flitorescein-EX, fluoresceinisothiocyanate, Rhodamine Red-X, Lissamine rhodamine B, Calcein,Fluorescein, Rhodamine, one or more BODIPY dyes, Texas Red, Oregon Green514, and one or more Alexa Fhiors. Representative BOD1PY dyes includeBODIPY FL, BODIPY R6G, BOD1PY TMR, BOD1PY 581/591, BODIPY TR, BODIPY630/650 and BODIPY 650/665. Representative Alexa Fluors include AlexaFluor 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633,635, 647, 660, 680, 700, 750 and 790. In exemplary aspects, thefluorescent label comprises one or more of Oregon Green 488,fluorescein-EX, FITC, Rhodamine Red-X, Lissamine rhodamine B, calcein,fluorescein, rhodamine, BODIPYS, and Texas Red, e.g. which are disclosedin Molecular Probes Handbook, 1 1th Edition (2010). In exemplaryaspects, the detectable label is selected from radioisotopes,chromophores, fluorophores, fluorochromes, enzymes (e.g., horseradishperoxidase), linker molecules or other moieties or compounds whicheither emit a detectable signal (e.g., radioactivity, fluorescence,color) or emit a detectable signal after exposure of the label to itssubstrate. A variety of detectable label/substrate pairs (e.g.,horseradish peroxidase/diaminobenzidine, biotin/streptavidin,luciferase/luciferin), methods for labeling antibodies, and methods forusing labeled secondary antibodies to detect an antigen are well knownin the art. See, e.g., Harlow and Lane, eds. (Using Antibodies: ALaboratory Manual (1999) Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.).

In some embodiments, the binding construct is directly joined to aheterologous moiety in the absence of a linker. In alternative aspects,the binding construct is indirectly connected to the heterologous moietyvia one or more linkers. Whether directly joined together or indirectlyjoined together through a linker, the binding construct may be connectedthrough covalent bonds (e.g., a peptide, ester, amide, or sulfhydrylbond) or non-covalent bonds (e.g., via hydrophobic interaction, hydrogenbond, van der Waals bond, electrostatic or ionic interaction), or acombination thereof. The binding construct of the invention andheterologous moiety may be connected via any means known in the art,including, but not limited to, via a linker of any of the invention.

Particular residues of the binding constructs described herein representexemplary sites at which a heterologous moiety may be attached. Forexample, Cys, His, Lys and N-terminal residues, Arg, Tyr, Asp, Glu, Ser,Thr, Pro represent sites at which the heterologous moiety may beattached. In some aspects, the residue (or a part thereof) is activatedwith one or more agents and/or chemicals prior to attaching theheterologous moiety.

Derivatization with bifunctional agents is useful for crosslinking thebinding construct to water-insoluble support matrixes. Such derivationmay also provide the linker that may connect adjacent binding elementsin a binding construct, or binding elements to a heterologous peptide,e.g., an Fc fragment. Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homo-bifunctional imidoesters, including disuccinimidyl esterssuch as 3,3′-dithiiobis(succinimidylpropioonate), and bifunctionalmaleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents suchas methyl-3-[(p-azidophenyl) dithio] propioimidate yieldphotoactivatable intermediates that are capable of forming cross linksin the presence of light. Alternatively, reactive water-insolublematrices such as cyanogen bromide-activated carbohydrates and thereactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016;4,195,128; 4,247,642; 4,229,537; and 4,330,440, incorporated herein byreference, are employed for protein immobilization.

In general, chemical derivatization may be performed under any suitablecondition used to react a protein with a heterologous moiety, e.g., anactivated polymer molecule. Methods for preparing chemical derivativesof polypeptides will generally comprise the steps of (a) reacting thebinding construct with the heterologous moiety, e.g., activated polymermolecule (such as a reactive ester or aldehyde derivative of the polymermolecule) under conditions whereby the binding construct becomesattached to one or more polymer molecules, and (b) obtaining thereaction product(s). The optimal reaction conditions will be determinedbased on known parameters and the desired result. For example, thelarger the ratio of polymer molecules:protein, the greater the amount ofattached polymer molecule. In some embodiments, the compound may have asingle polymer molecule moiety at the amino terminus. (See, e.g., U.S.Pat. No. 5,234,784).

Derivatized binding constructs disclosed herein may have additionalactivities, enhanced or reduced biological activity, or othercharacteristics, such as increased or decreased half-life, as comparedto the non-derivatized molecules.

Conjugates: Fc Fusions

For substituents such as an Fc region of human IgG, the fusion can befused directly to a binding construct of the invention or fused throughan intervening sequence. For example, a human IgG hinge, CH2 and CH3region may be fused at either the N-terminus or C-terminus of a bindingconstruct to attach the Fc region. The resulting Fc-fusion constructenables purification via a Protein A affinity column (Pierce, Rockford,Ill.). Peptide and proteins fused to an Fc region can exhibit asubstantially greater half-life in vivo than the unfused counterpart. Afusion to an Fc region allows for dimerization/multimerization of thefusion polypeptide. The Fc region may be a naturally occurring Fcregion, or may be modified for superior characteristics, e.g.,therapeutic or diagnostic qualities, circulation time, reducedaggregation. As noted above, in some embodiments, the binding constructsare conjugated, e.g., fused to an immunoglobulin or portion thereof(e.g., variable region, CDR, or Fc region). Known types ofimmunoglobulins (Ig) include IgG, IgA, IgE, IgD or IgM. The Fc region isa C-terminal region of an Ig heavy chain, which is responsible forbinding to Fc receptors that carry out activities such as recycling(which results in prolonged half-life), antibody dependent cell-mediatedcytotoxicity (ADCC), and complement dependent cytotoxicity (CDC).

For example, according to some definitions the human IgG heavy chain Fcregion stretches from Cys226 to the C-terminus of the heavy chain. The“hinge region” generally extends from Glu216 to Pro230 of human IgG1(hinge regions of other IgG isotypes may be aligned with the IgG1sequence by aligning the cysteines involved in cysteine bonding). The Fcregion of an IgG includes two constant domains, CH2 and CH3. The CH2domain of a human IgG Fc region usually extends from amino acids 231 toamino acid 341. The CH3 domain of a human IgG Fc region usually extendsfrom amino acids 342 to 447. References made to amino acid numbering ofimmunoglobulins or immunoglobulin fragments, or regions, are all basedon Kabat et al. 1991, Sequences of Proteins of Immunological Interest,U.S. Department of Public Health, Bethesda, Md. In related embodiments,the Fc region may comprise one or more native or modified constantregions from an immunoglobulin heavy chain, other than CH1, for example,the CH2 and CH3 regions of IgG and IgA, or the CH3 and CH4 regions ofIgE.

Suitable heterologous moieties include portions of immunoglobulinsequence that include the FcRn binding site. FcRn, a salvage receptor,is responsible for recycling immunoglobulins and returning them tocirculation in blood. The region of the Fc portion of IgG that binds tothe FcRn receptor has been described based on X-ray crystallography(Burmeister et al. 1994, Nature 372:379). The major contact area of theFc with the FcRn is near the junction of the CH2 and CH3 domains.Fc-FcRn contacts are all within a single Ig heavy chain. The majorcontact sites include amino acid residues 248, 250-257, 272, 285, 288,290-291, 308-311, and 314 of the CH2 domain and amino acid residues385-387, 428, and 433-436 of the CH3 domain.

Amino acid modifications may be made to the Fc region of animmunoglobulin. Such variant Fc regions comprise at least one amino acidmodification in the CH3 domain of the Fc region (residues 342-447)and/or at least one amino acid modification in the CH2 domain of the Fcregion (residues 231-341). Mutations believed to impart an increasedaffinity for FcRn include T256A, T307A, E380A, and N434A (Shields et al.2001, J. Biol. Chem. 276:6591). Other mutations may reduce binding ofthe Fc region to FcγRI, FcγRIIA, FcγRIIB, and/or FcγRIIIA withoutsignificantly reducing affinity for FcRn. For example, substitution ofthe Asn at position 297 of the Fc region with Ala or another amino acidremoves a highly conserved N-glycosylation site and may result inreduced immunogenicity with concomitant prolonged half-life of the Fcregion, as well as reduced binding to FcγRs (Routledge et al. 1995,Transplantation 60:847; Friend et al. 1999, Transplantation 68:1632;Shields et al. 1995, J. Biol. Chem. 276:6591). Amino acid modificationsat positions 233-236 of IgG1 have been made that reduce binding to FcγRs(Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al.1999, Eur. J. Immunol. 29:2613). Some exemplary amino acid substitutionsare described in U.S. Pat. Nos. 7,355,008 and 7,381,408, eachincorporated by reference herein in its entirety.

Heterologous Moieties: Polymers, Carbohydrates, and Lipids

In exemplary embodiments, the heterologous moiety is a polymer. Thepolymer may be branched or unbranched. The polymer may be of anymolecular weight. The polymer in some embodiments has an averagemolecular weight of between about 2 kDa to about 100 kDa (the term“about” indicating that in preparations of a water soluble polymer, somemolecules will weigh more, some less, than the stated molecular weight).The average molecular weight of the polymer is in some aspect betweenabout 5 kDa and about 50 kDa, between about 12 kDa to about 40 kDa orbetween about 20 kDa to about 35 kDa.

In some embodiments, the polymer is modified to have a single reactivegroup, such as an active ester for acylation or an aldehyde foralkylation, so that the degree of polymerization may be controlled. Thepolymer in some embodiments is water soluble so that the protein towhich it is attached does not precipitate in an aqueous environment,such as a physiological environment. In some embodiments, when, forexample, the composition is used for therapeutic use, the polymer ispharmaceutically acceptable. Additionally, in some aspects, the polymeris a mixture of polymers, e.g., a co-polymer, a block co-polymer.

In some embodiments, the polymer is selected from the group consistingof: polyamides, polycarbonates, polyalkylenes and derivatives thereofincluding, polyalkylene glycols, polyalkylene oxides, polyalkyleneterepthalates, polymers of acrylic and methacrylic esters, includingpoly(methyl methacrylate), poly(ethyl methacrylate),poly(butylmethacrylate), poly(isobutyl methacrylate),poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecylacrylate), polyvinyl polymers including polyvinyl alcohols, polyvinylethers, polyvinyl esters, polyvinyl halides, poly(vinyl acetate), andpolyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes andco-polymers thereof, celluloses including alkyl cellulose, hydroxyalkylcelluloses, cellulose ethers, cellulose esters, nitro celluloses, methylcellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propylmethyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate,cellulose propionate, cellulose acetate butyrate, cellulose acetatephthalate, carboxylethyl cellulose, cellulose triacetate, and cellulosesulphate sodium salt, polypropylene, polyethylenes includingpoly(ethylene glycol), poly(ethylene oxide), and poly(ethyleneterephthalate), and polystyrene.

In some aspects, the polymer is a biodegradable polymer, including asynthetic biodegradable polymer (e.g., polymers of lactic acid andglycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes,poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone)),and a natural biodegradable polymer (e.g., alginate and otherpolysaccharides including dextran and cellulose, collagen, chemicalderivatives thereof (substitutions, additions of chemical groups, forexample, alkyl, alkylene, hydroxylations, oxidations, and othermodifications routinely made by those skilled in the art), albumin andother hydrophilic proteins (e.g., zein and other prolamines andhydrophobic proteins)), as well as any copolymer or mixture thereof. Ingeneral, these materials degrade either by enzymatic hydrolysis orexposure to water in vivo, by surface or bulk erosion.

In some aspects, the polymer is a bioadhesive polymer, such as abioerodible hydrogel described by H. S. Sawhney, C. P. Pathak and J. A.Hubbell in Macromolecules, 1993, 26, 581-587, the teachings of which areincorporated herein, polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethyl methacrylates), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate).

In some embodiments, the polymer is a water-soluble polymer or ahydrophilic polymer. Suitable water-soluble polymers are known in theart and include, for example, polyvinylpyrrolidone, hydroxypropylcellulose (HPC; Klucel), hydroxypropyl methylcellulose (HPMC; Methocel),nitrocellulose, hydroxypropyl ethylcellulose, hydroxypropylbutylcellulose, hydroxypropyl pentylcellulose, methyl cellulose,ethylcellulose (Ethocel), hydroxyethyl cellulose, various alkylcelluloses and hydroxyalkyl celluloses, various cellulose ethers,cellulose acetate, carboxymethyl cellulose, sodium carboxymethylcellulose, calcium carboxymethyl cellulose, vinyl acetate/crotonic acidcopolymers, poly-hydroxyalkyl methacrylate, hydroxymethyl methacrylate,methacrylic acid copolymers, polymethacrylic acid,polymethylmethacrylate, maleic anhydride/methyl vinyl ether copolymers,poly vinyl alcohol, sodium and calcium polyacrylic acid, polyacrylicacid, acidic carboxy polymers, carboxypolymethylene, carboxyvinylpolymers, polyoxyethylene polyoxypropylene copolymer,polymethylvinylether co-maleic anhydride, carboxymethylamide, potassiummethacrylate divinylbenzene co-polymer, polyoxyethyleneglycols,polyethylene oxide, and derivatives, salts, and combinations thereof. Insome aspects, the water soluble polymers or mixtures thereof include,but are not limited to, N-linked or O-linked carbohydrates, sugars,phosphates, carbohydrates; sugars; phosphates; polyethylene glycol (PEG)(including the forms of PEG that have been used to derivatize proteins,including mono-(C1-C10) alkoxy- or aryloxy-polyethylene glycol);monomethoxy-polyethylene glycol; dextran (such as low molecular weightdextran, of, for example about 6 kD), cellulose; cellulose; othercarbohydrate-based polymers, poly-(N-vinyl pyrrolidone)polyethyleneglycol, propylene glycol homopolymers, a polypropylene oxide/ethyleneoxide co-polymer, polyoxyethylated polyols (e.g., glycerol) andpolyvinyl alcohol. Also encompassed by the present invention arebifunctional crosslinking molecules which may be used to preparecovalently attached multimers.

A particularly preferred water-soluble polymer for use herein ispolyethylene glycol (PEG). As used herein, polyethylene glycol is meantto encompass any of the forms of PEG that can be used to derivatizeother proteins, such as mono-(C1-C10) alkoxy- or aryloxy-polyethyleneglycol. PEG is a linear or branched neutral polyether, available in abroad range of molecular weights, and is soluble in water and mostorganic solvents. PEG is effective at excluding other polymers orpeptides when present in water, primarily through its high dynamic chainmobility and hydrophibic nature, thus creating a water shell orhydration sphere when attached to other proteins or polymer surfaces.PEG is nontoxic, non-immunogenic, and approved by the Food and DrugAdministration for internal consumption.

Methods for preparing pegylated compounds may comprise the steps of (a)reacting the compound with polyethylene glycol (such as a reactive esteror aldehyde derivative of PEG) under conditions whereby the compoundbecomes attached to one or more PEG groups, and (b) obtaining thereaction product(s). In general, the optimal reaction conditions for theacylation reactions will be determined based on known parameters and thedesired result. For example, the larger the ratio of PEG:compound, thegreater the percentage of poly-pegylated product. In some embodiments,the binding construct will have a single PEG moiety at the N-terminus.See U.S. Pat. No. 8,234,784, herein incorporated by reference.

In some embodiments, the heterologous moiety is a carbohydrate. In someembodiments, the carbohydrate is a monosaccharide (e.g., glucose,galactose, fructose), a disaccharide (e.g., sucrose, lactose, maltose),an oligosaccharide (e.g., raffinose, stachyose), a polysaccharide (astarch, amylase, amylopectin, cellulose, chitin, callose, laminarin,xylan, mannan, fucoidan, galactomannan.

In some embodiments, the heterologous moiety is a lipid. The lipid, insome embodiments, is a fatty acid, eicosanoid, prostaglandin,leukotriene, thromboxane, N-acyl ethanolamine), glycerolipid (e.g.,mono-, di-, tri-substituted glycerols), glycerophospholipid (e.g.,phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine,phosphatidylserine), sphingolipid (e.g., sphingosine, ceramide), sterollipid (e.g., steroid, cholesterol), prenol lipid, saccharolipid, or apolyketide, oil, wax, cholesterol, sterol, fat-soluble vitamin,monoglyceride, diglyceride, triglyceride, a phospholipid.

Heterologous Moieties: Therapeutic Agents

In some embodiments, the heterologous moiety is a therapeutic agent. Thetherapeutic agent may be any of those known in the art. Examples oftherapeutic agents that are contemplated herein include, but are notlimited to, natural enzymes, proteins derived from natural sources,recombinant proteins, natural peptides, synthetic peptides, cyclicpeptides, antibodies, receptor agonists, cytotoxic agents,immunoglobins, beta-adrenergic blocking agents, calcium channelblockers, coronary vasodilators, cardiac glycosides, antiarrhythmics,cardiac sympathomemetics, angiotensin converting enzyme (ACE)inhibitors, diuretics, inotropes, cholesterol and triglyceride reducers,bile acid sequestrants, fibrates, 3-hydroxy-3-methylgluteryl (HMG)-CoAreductase inhibitors, niacin derivatives, antiadrenergic agents,alpha-adrenergic blocking agents, centrally acting antiadrenergicagents, vasodilators, potassium-sparing agents, thiazides and relatedagents, angiotensin II receptor antagonists, peripheral vasodilators,antiandrogens, estrogens, antibiotics, retinoids, insulins and analogs,alpha-glucosidase inhibitors, biguanides, meglitinides, sulfonylureas,thizaolidinediones, androgens, progestogens, bone metabolism regulators,anterior pituitary hormones, hypothalamic hormones, posterior pituitaryhormones, gonadotropins, gonadotropin-releasing hormone antagonists,ovulation stimulants, selective estrogen receptor modulators,antithyroid agents, thyroid hormones, bulk forming agents, laxatives,antiperistaltics, flora modifiers, intestinal adsorbents, intestinalanti-infectives, antianorexic, anticachexic, antibulimics, appetitesuppressants, antiobesity agents, antacids, upper gastrointestinal tractagents, anticholinergic agents, aminosalicylic acid derivatives,biological response modifiers, corticosteroids, antispasmodics, 5-HT4partial agonists, antihistamines, cannabinoids, dopamine antagonists,serotonin antagonists, cytoprotectives, histamine H2-receptorantagonists, mucosal protective agent, proton pump inhibitors, H. pylorieradication therapy, erythropoieses stimulants, hematopoietic agents,anemia agents, heparins, antifibrinolytics, hemostatics, bloodcoagulation factors, adenosine diphosphate inhibitors, glycoproteinreceptor inhibitors, fibrinogen-platelet binding inhibitors,thromboxane-A₂ inhibitors, plasminogen activators, antithromboticagents, glucocorticoids, mineralcorticoids, corticosteroids, selectiveimmunosuppressive agents, antifungals, drugs involved in prophylactictherapy, AIDS-associated infections, cytomegalovirus, non-nucleosidereverse transcriptase inhibitors, nucleoside analog reverse transcriptseinhibitors, protease inhibitors, anemia, Kaposi's sarcoma, aminoglycosides, carbapenems, cephalosporins, glycopoptides, lincosamides, macrolies, oxazolidinones, penicillins, streptogram ins,sulfonamides, trimethoprim and derivatives, tetracyclines,anthelmintics, amebicies, biguanides, cinchona alkaloids, folic acidantagonists, quinoline derivatives, Pneumocystis carinii therapy,hydrazides, imidazoles, triazoles, nitroimidzaoles, cyclic amines,neuraminidase inhibitors, nucleosides, phosphate binders, cholinesteraseinhibitors, adjunctive therapy, barbiturates and derivatives,benzodiazepines, gamma aminobutyric acid derivatives, hydantoinderivatives, iminostilbene derivatives, succinimide derivatives,anticonvulsants, ergot alkaloids, antimigrane preparations, biologicalresponse modifiers, carbamic acid eaters, tricyclic derivatives,depolarizing agents, nondepolarizing agents, neuromuscular paralyticagents, CNS stimulants, dopaminergic reagents, monoamine oxidaseinhibitors, COMT inhibitors, alkyl sulphonates, ethylenimines,imidazotetrazines, nitrogen mustard analogs, nitrosoureas,platinum-containing compounds, antimetabolites, purine analogs,pyrimidine analogs, urea derivatives, antracyclines, actinomycinds,camptothecin derivatives, epipodophyllotoxins, taxanes, vinca alkaloidsand analogs, antiandrogens, antiestrogens, nonsteroidal aromataseinhibitors, protein kinase inhibitor antineoplastics,azaspirodecanedione derivatives, anxiolytics, stimulants, monoamindreuptake inhibitors, selective serotonin reuptake inhibitors,antidepressants, benzisooxazole derivatives, butyrophenone derivatives,dibenzodiazepine derivatives, dibenzothiazepine derivatives,diphenylbutylpiperidine derivatives, phenothiazines,thienobenzodiazepine derivatives, thioxanthene derivatives, allergenicextracts, nonsteroidal agents, leukotriene receptor antagonists,xanthines, endothelin receptor antagonist, prostaglandins, lungsurfactants, mucolytics, antimitotics, uricosurics, xanthine oxidaseinhibitors, phosphodiesterase inhibitors, metheamine salts, nitrofuranderivatives, quinolones, smooth muscle relaxants, parasympathomimeticagents, halogenated hydrocarbons, esters of amino benzoic acid, amides(e.g. lidocaine, articaine hydrochloride, bupivacaine hydrochloride),antipyretics, hynotics and sedatives, cyclopyrrolones,pyrazolopyrimidines, nonsteroidal anti-inflammatory drugs, opioids,para-aminophenol derivatives, alcohol dehydrogenase inhibitor, heparinantagonists, adsorbents, emetics, opioid antagonists, cholinesterasereactivators, nicotine replacement therapy, vitamin A analogs andantagonists, vitamin B analogs and antagonists, vitamin C analogs andantagonists, vitamin D analogs and antagonists, vitamin E analogs andantagonists, vitamin K analogs and antagonists.

Conjugates: Detecting Agents

In exemplary embodiments, the binding construct is conjugated to adetecting agent. In exemplary embodiments, the detecting agent iscapable of emitting a detectable (measurable) signal based on enzymaticactivity, radioactivity, chromogenic activity, and/or binding activity.In exemplary embodiments, the signal is radioactive, chromogenic,colorimetric, fluorometric, chemiluminescent, enhanced chemiluminescent,direct fluorescent, time-resolved fluorescent, direct chemiluminescent,phosphorescent, enzymatic, or based on binding of a micro- ornanoparticle, streptavidin/avidin-biotin and protein A. In exemplaryembodiments, the detecting agent comprises an enzyme, a radioactiveisotope, a DNA reporter, a chromogenic or fluorogenic reporter, or anelectrochemiluminescent tag. In exemplary aspects, the enzyme ishorseradish peroxidase (HRP), alkaline phosphatase (AP), glucoseoxidase, or beta-galactosidase. In exemplary aspects, the enzyme whenexposed to certain reagents cause chemiluminescence or light production.In exemplary aspects, the radioisotope is I¹²⁵. In exemplary aspects,the DNA reporter is a DNA probe. In exemplary aspects, the fluorogenicreporter is phycoerythrin (PE), e.g., B-PE, R-PE, or allophycocyanin(APC).

Conjugates: Dimers & Multimers

In some embodiments, the binding construct is provided as a dimer or amultimer in which more than one binding construct of the invention arelinked together. The dimer in some aspects is a homodimer comprising twobinding constructs of the same type (e.g., same structure) linkedtogether. In alternative aspects, the dimer is a heterodimer comprisingtwo binding constructs of the invention, wherein the two bindingconstructs are structurally distinct from each other. The multimer insome aspects is a homomultimer comprising more than one bindingconstruct of the invention and each binding construct is of the sametype (e.g., same structure). In alternative aspects, the multimer is aheteromultimer comprising more than one binding construct of theinvention and wherein at least two binding constructs of theheteromultimer are structurally distinct from the other. In exemplaryaspects, the binding construct comprises a dimer, e.g., a homodimer, oftwo Fab fragments, each Fab fragment of which binds to FIXp and not toWT FIX, e.g., binds to FIXp even in the presence of WT FIX or in asample comprising human plasma. In exemplary aspects, the homodimercomprising two Fab fragments is bivalent yet monospecific for FIXp. Inexemplary aspects, each Fab fragment of the homodimer comprises theamino acid sequence of SEQ ID NOs: 6-11. In exemplary aspects, one ormore amino acids are present between each of SEQ ID NOs: 6-11. Inexemplary aspects, each Fab fragment of the homodimer comprises thesequence of SEQ ID NO: 24 or SEQ ID NO: 25 or both SEQ ID NOs: 24 and25. In exemplary aspects, each Fab fragment of the homodimer comprisesthe sequence of SEQ ID NO: 26 or SEQ ID NO: 27 or both SEQ ID NOs: 26and 27.

Two or more of the binding constructs can be linked together usingstandard linking agents and procedures known to those skilled in theart. In certain embodiments, the linker connecting the two (or more)binding constructs is a linker known in the art. In some embodiments,the linker is a disulfide bond. For example, each monomer of the dimermay comprise a sulfhydryl and the sulfur atom of each participates inthe formation of the disulfide bond. In some embodiments, the linker isa helix-turn-helix motif. In exemplary aspects, each monomer of thedimer is connected via a helix-turn-helix motif. In exemplary aspects,each monomer of the dimer is connected via an alkaline phosphatasedomain.

In exemplary aspects, the homodimer comprising two Fab fragmentscomprises a linker connecting the two Fab fragments. In exemplaryaspects, the homodimer comprises a synthetic double helix loop helixmotif, such as that described in Haylock et al., Int J. Oncol. 48(2):461-470 (2016) or Wang et al., Anal. Chem. 78: 997-1004 (2006). Inexemplary aspects, the homodimer comprises a constant antibody domain.Such antibody domains are described in Hu et al., Cancer Res 56:3055-3061 (1996) and McGregor et al., Mol Immuno 31: 219-226 (1994). Inexemplary aspects, the homodimer comprises a bacterial alkalinephosphatase domain, such as that described in Wang et al. (2006), supra.In exemplary aspects, the homodimer comprises the sequence of SEQ ID NO:28 or SEQ ID NO: 27 or both SEQ ID NOs: 28 and 27.

Nucleic Acids

Further provided herein are nucleic acids comprising a nucleotidesequence encoding any of the binding constructs (e.g., antibodies,antigen-binding fragments, polypeptides, or conjugates) describedherein. By “nucleic acid” as used herein includes “polynucleotide,”“oligonucleotide,” and “nucleic acid molecule,” and generally means apolymer of DNA or RNA, which can be single-stranded or double-stranded,synthesized or obtained (e.g., isolated and/or purified) from naturalsources, which can contain natural, non-natural or altered nucleotides,and which can contain a natural, non-natural or altered inter-nucleotidelinkage, such as a phosphoroamidate linkage or a phosphorothioatelinkage, instead of the phosphodiester found between the nucleotides ofan unmodified oligonucleotide. It is generally preferred that thenucleic acid does not comprise any insertions, deletions, inversions,and/or substitutions. However, it may be suitable in some instances, asdiscussed herein, for the nucleic acid to comprise one or moreinsertions, deletions, inversions, and/or substitutions.

Preferably, the nucleic acids of the invention are recombinant. As usedherein, the term “recombinant” refers to (i) molecules that areconstructed outside living cells by joining natural or synthetic nucleicacid segments to nucleic acid molecules that can replicate in a livingcell, or (ii) molecules that result from the replication of thosedescribed in (i) above. For purposes herein, the replication can be invitro replication or in vivo replication.

The nucleic acids can be constructed based on chemical synthesis and/orenzymatic ligation reactions using procedures known in the art. See, forexample, Sambrook et al., supra, and Ausubel et al., supra. For example,a nucleic acid can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed upon hybridization (e.g.,phosphorothioate derivatives and acridine substituted nucleotides).Examples of modified nucleotides that can be used to generate thenucleic acids include, but are not limited to, 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil,5-carboxymethylaminomethyl-2-thiouridme,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N-substitutedadenine, 7-methylguanine, 5-methylammomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouratil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleicacids of the invention can be purchased from companies, such asMacromolecular Resources (Fort Collins, Colo.) and Synthegen (Houston,Tex.).

In some aspects, the nucleic acid encodes only a portion of theantibodies, antigen-binding fragments, polypeptides, or conjugates. Forexample, when the conjugate comprises a polymer, which does not compriseamino acids and thus is not encoded by a nucleic acid, the nucleic acidencodes only the part of the conjugate which can be encoded by a nucleicacid. In exemplary embodiments, the nucleic acid comprises a nucleotidesequence encoding a polypeptide comprising an amino acid sequencecomprising each of SEQ ID NOs: 6-11. In exemplary aspects, the nucleicacid encodes the polypeptide comprising an amino acid sequencecomprising each of SEQ ID NOs: 6-11 wherein one or more amino acids arepresent between each of SEQ ID NOs: 6-11. In exemplary aspects, thenucleic acid encodes the polypeptide which further comprises anexpression tag, e.g., a FLAG tag comprising DYKDDDDK (SEQ ID NO: 12)and/or a hexa-His tag comprising HHHHHH (SEQ ID NO: 13).

The nucleic acids are useful in e.g., methods of recombinant productionof the binding constructs of the invention.

Recombinant Expression Vector

The nucleic acids of the invention can be incorporated into arecombinant expression vector, or “vector”. In this regard, theinvention provides recombinant expression vectors or “vectors”comprising any of the nucleic acids of the invention. For purposesherein, the term “recombinant expression vector” or “vector” means agenetically-modified oligonucleotide or polynucleotide construct thatpermits the expression of an mRNA, protein, polypeptide, or peptide by ahost cell, when the construct comprises a nucleotide sequence encodingthe mRNA, protein, polypeptide, or peptide, and the vector is contactedwith the cell under conditions sufficient to have the mRNA, protein,polypeptide, or peptide expressed within the cell. The vectors of theinvention are not naturally-occurring as a whole. However, parts of thevectors can be naturally-occurring. The inventive recombinant expressionvectors can comprise any type of nucleotides, including, but not limitedto DNA and RNA, which can be single-stranded or double-stranded,synthesized or obtained in part from natural sources, and which cancontain natural, non-natural or altered nucleotides. The recombinantexpression vectors can comprise naturally-occurring ornon-naturally-occurring internucleotide linkages, or both types oflinkages. Preferably, the altered nucleotides or non-naturally occurringinternucleotide linkages do not hinder the transcription or replicationof the vector.

The recombinant expression vector of the invention can be any suitablerecombinant expression vector, and can be used to transform or transfectany suitable host. Suitable vectors include those designed forpropagation and expansion or for expression or both, such as plasm idsand viruses. The vector can be selected from the group consisting of thepUC series (Fermentas Life Sciences), the pBluescript series(Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.),the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series(Clontech, Palo Alto, Calif.). Bacteriophage vectors, such as AGTIO,AGTI 1, AZapll (Stratagene), AEMBL4, and ANMI 149, also can be used.Examples of plant expression vectors include pBIO1, pBI101.2, pBI101.3,pBI121 and pBIN19 (Clontech). Examples of animal expression vectorsinclude pEUK-CI, pMAM and pMAMneo (Clontech). Preferably, therecombinant expression vector is a viral vector, e.g., a retroviralvector.

The recombinant expression vectors of the invention can be preparedusing standard recombinant DNA techniques described in, for example,Sambrook et al., supra, and Ausubel et al., supra. Constructs ofexpression vectors, which are circular or linear, can be prepared tocontain a replication system functional in a prokaryotic or eukaryotichost cell. Replication systems can be derived, e.g., from ColEl, 2 pplasmid, A, SV40, bovine papilloma virus, and the like.

Desirably, the recombinant expression vector comprises regulatorysequences, such as transcription and translation initiation andtermination codons, which are specific to the type of host (e.g.,bacterium, fungus, plant, or animal) into which the vector is to beintroduced, as appropriate and taking into consideration whether thevector is DNA- or RNA-based.

The recombinant expression vector can comprise a native or normativepromoter operably linked to the nucleotide sequence encoding thepolypeptide (including functional portions and functional variantsthereof), or to the nucleotide sequence which is complementary to orwhich hybridizes to the nucleotide sequence encoding the polypeptide.The selection of promoters, e.g., strong, weak, inducible,tissue-specific and developmental-specific, is within the ordinary skillof the artisan. Similarly, the combining of a nucleotide sequence with apromoter is also within the skill of the artisan. The promoter can be anon-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV)promoter, an SV40 promoter, an RSV promoter, and a promoter found in thelong-terminal repeat of the murine stem cell virus.

The inventive recombinant expression vectors can be designed for eithertransient expression, for stable expression, or for both. Also, therecombinant expression vectors can be made for constitutive expressionor for inducible expression. Further, the recombinant expression vectorscan be made to include a suicide gene.

Host Cells

The invention further provides a host cell comprising any of the nucleicacids or recombinant expression vectors described herein. As usedherein, the term “host cell” refers to any type of cell that can containand express the inventive recombinant expression vector. The host cellcan be a eukaryotic cell, e.g., plant, animal, fungi, or algae, or canbe a prokaryotic cell, e.g., bacteria or protozoa. The host cell can bea cultured cell or a primary cell, i.e., isolated directly from anorganism, e.g., a human. The host cell can be an adherent cell or asuspended cell, i.e., a cell that grows in suspension. Suitable hostcells are known in the art and include, for instance, DH5a E. colicells, Chinese hamster ovarian cells, monkey VERO cells, COS cells,HEK293 cells, and the like. For purposes of amplifying or replicatingthe recombinant expression vector, the host cell is preferably aprokaryotic cell, e.g., a DH5a cell. For purposes of producing arecombinant polypeptide the host cell is preferably a mammalian cell,e.g., a CHO cell.

Kits

Provided herein are kits comprising any one or more of the bindingconstructs of the present disclosure. In exemplary embodiments, the kitcomprises an antibody or antigen-binding fragment or polypeptide orconjugate or nucleic acid or vector or host cell, as described herein ora combination of any of the foregoing. In exemplary aspects, the bindingconstruct is provided in the kit in a predetermined amount orconcentration. For example, the kit may be a detection kit comprising apredetermined amount of the binding construct for detecting FIX Padua ina sample. In exemplary embodiments, the one or more of the bindingconstructs of the present disclosure is provided in the kit in anaqueous solution. In exemplary aspects, the aqueous solution is providedto the end-user on dry ice. In some aspects, the aqueous solution isprovided to the end-user separately from the other components of thekit. In exemplary embodiments, the binding constructs of the presentdisclosure are provided in the kit in a lyophilized or otherfreeze-dried form. In exemplary aspects, the binding constructs of thepresent disclosures are provided in the kit in a frozen or cryopreservedform. In exemplary aspects, the concentration of the antibody orantigen-binding fragment or polypeptide or conjugate provided in the kitis about 1-10 μg/mL or about 1-5 μg/mL. In exemplary aspects, theconcentration of the antibody or antigen-binding fragment or polypeptideor conjugate provided in the kit is about 1.5 μg/mL to about 2.0 μg/m L.

In exemplary aspects, the kit comprises a solid support, and inexemplary aspects the antibody or antigen-binding fragment orpolypeptide or conjugate is pre-coated onto the solid support. Inexemplary aspects, the kit comprises a solid support selected from thegroup consisting of a tube, a dish, a flask, a bag, a plate (e.g., amicrotiter plate), a membrane, a filter, a bead, a fiber, a probe, andthe like. In exemplary aspects, the solid support is made of a polymer.In exemplary aspects, the solid support is made of agarose, cellulose,dextran, polyacrylamide, latex, or controlled pore glass. In exemplaryaspects, the solid support is made of agarose. In exemplary aspects, thesolid support is made of polyvinyl difluoride (PVDF), nitrocellulose,nylon 66, protran nitrocellulose, or paper. In exemplary aspects, themembrane is one of the Immobilon®, Protran®, QuickDraw®, Westran®,Whatman® or Hybond® membranes (Sigma-Aldrich, St. Louis, Mo.). Inexemplary aspects, the solid support is a polymer bead, a microtiterplate, a membrane or a filter. In exemplary aspects, the kit comprises asolid support pre-coated with a solution comprising about 100 ng ormore, about 150 ng or more, about 200 ng or more, about 500 ng or moreof the antibody or antigen-binding fragment or polypeptide or conjugate.In certain aspects, the kit comprises a solid support pre-coated with asolution comprising about 50 ng to about 550 ng, about 100 ng to about500 ng, about 125 ng to about 400 ng, about 150 ng to about 350 ng,about 175 ng to about 300 ng, or about 200 ng to about 250 ng of theantibody or antigen-binding fragment or polypeptide or conjugate. Incertain aspects, the kit comprises a solid support pre-coated with asolution comprising about 100 ng to about 150 ng, about 150 ng to about200 ng, about 200 ng to about 500 ng of the antibody or antigen-bindingfragment or polypeptide or conjugate. In exemplary aspects, the kitcomprises a solid support comprising pre-aliquoted amounts of theantibody or antigen-binding fragment or polypeptide or conjugate. Inexemplary aspects, the kit comprises a microtiter plate, wherein eachwell of the microtiter plate comprises a solution comprising about 100μL to about 500 μL of a solution comprising about 1-10 μg/mL or about1-5 μg/mL of the antibody or antigen-binding fragment or polypeptide orconjugate. In exemplary aspects, the kit comprises a microtiter plate,wherein each well of the microtiter plate comprises a solutioncomprising about 100 μL to about 500 μL of a solution comprising about2.5 μg/mL of the antibody or antigen-binding fragment or polypeptide orconjugate.

In exemplary aspects, the kit comprises additional reagents, substrates,solvents, buffers, diluents, etc., used in the detection methodsdescribed herein. In exemplary aspects, any one or more of theadditional components are provided in the kit in a predetermined amount,e.g., the amount necessary and suitable for a detection assay. Inexemplary aspects, the kit comprises a secondary antibody which binds tothe FIX Padua-binding antibody, antigen-binding fragment, polypeptide orconjugate. In exemplary aspects, the secondary antibody comprising adetecting agent. In exemplary embodiments, the detecting agent comprisesan enzyme, a radioactive isotope, a DNA reporter, a chromogenic orfluorogenic reporter, or an electrochemiluminescent tag. The detectingagent can be any of the detecting agents described herein. In exemplaryaspects, the secondary antibody or the FIX Padua-binding antibody,antigen-binding fragment, polypeptide or conjugate is attached to adetecting agent.

Compositions

Provided herein are compositions comprising any one or more of thebinding constructs of the present disclosure. In exemplary aspects, thecomposition comprises a binding construct as described herein admixedwith a detecting agent. In exemplary aspects, the detecting agent is anydetecting agent described herein. See the section entitled “Conjugates:Detecting Agents”.

In exemplary aspects, the composition comprises a binding construct asdescribed herein admixed with a biological sample obtained from asubject. In exemplary aspects, the biological sample is any biologicalsample described herein. See the section entitled “Samples”. Inexemplary aspects, the composition comprises an antibody orantigen-binding fragment or polypeptide or conjugate, as describedherein, admixed with a biological sample comprising human plasma, or adiluted fraction thereof. In exemplary aspects, the compositioncomprises an antibody or antigen-binding fragment or polypeptide orconjugate, as described herein, admixed with a biological samplecomprising human tissue, or cells thereof. In exemplary aspects, thebiological sample comprises liver tissue. In exemplary aspects, thecomposition further comprises a detecting agent.

Compositions comprising any one or more of the binding constructs of thepresent disclosure admixed with a sample, e.g., a biological sample,comprising human plasma proteins is further provided herein. Inexemplary aspects, the composition comprises an antibody orantigen-binding fragment or polypeptide or conjugate, as describedherein, admixed with at least one of the human plasma proteins selectedfrom the group consisting of Factor IX, Factor II, and Factor X, andvariants thereof. In exemplary aspects, the composition furthercomprises a detecting agent.

Detection Methods

Binding constructs provided herein are useful in, e.g., detectionmethods that allow for unambiguous or specific detection of FIX Padua insamples, e.g., clinical samples comprising, e.g., FIX Padua and WT FIX.The binding constructs can be used in any antibody-based assay ortechnique or any immunoassay known in the art, such as, but not limitedto, radioimmunoassay (RIA), magnetic immunoassay (MIA),immunocytochemical (ICC) assays, immunohistochemical (IHC) assays,immunofluorescent assays, ELISA, EIA, ELISPOT, enzyme multipliedimmunoassay, radiobinding assay, Western blotting, immunoprecipitation,dot blots, flow cytometry, real-time immunoquantitative PCR, proteinmicroarrays and the like. See, e.g., The Immunoassay Handbook (FourthEdition); Theory and Applications of Ligand Binding, ELISA and RelatedTechniques, ed. Wild, Elsevier Ltd. (Oxford, UK) 2013, Green andSambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold SpringHarbor Laboratory Press (Cold Spring Harbor, N.Y.) 2012, andImmunoassay, Diamandis and Christopolous, Academic Press 1996.

Accordingly, provided herein are uses of the binding construct (e.g.,antibody or antigen-binding fragment, polypeptide, or conjugate),nucleic acid, vector, host cell, and/or kit described herein fordetecting Factor IX Padua in a sample. In exemplary aspects, the sampleis a biological sample that has been obtained from a subject who hasbeen administered an expression vector comprising a nucleic acidencoding FIX Padua. For example, in various embodiments, the subject issuffering from a bleeding disorder and is undergoing Factor IXreplacement therapy, optionally achieved by expression of a nucleic acidcomprising a nucleotide sequence encoding a heterologous Factor IXPadua.

Also provided herein are methods of detecting Factor IX Padua in asample obtained from a subject. In exemplary embodiments, the methodcomprises (i) contacting the sample with a binding construct (e.g., anantibody or antigen-binding fragment or polypeptide or conjugate) asdescribed herein to form a complex (e.g., an immunocomplex) comprisingFIX Padua and the binding construct (e.g., antibody, antigen-bindingfragment, polypeptide, or conjugate), and (ii) detecting the complex.

In exemplary embodiments, the FIX Padua comprises the amino acidsequence of SEQ ID NO: 1.

In exemplary embodiments, detecting the complex comprises detecting asignal of a detecting agent. In exemplary embodiments, the signal isbased on enzymatic activity, radioactivity, chromogenic activity, and/orbinding activity. In exemplary embodiments, the signal is radioactive,chromogenic, colorimetric, fluorometric, chemiluminescent, enhancedchemiluminescent, direct fluorescent, time-resolved fluorescent, directchemiluminescent, phosphorescent, enzymatic, or based on binding of amicro- or nanoparticle, streptavidin/avidin-biotin and protein A. Inexemplary embodiments, the detecting agent comprises an enzyme, aradioactive isotope, a DNA reporter, a chromogenic or fluorogenicreporter, an electrochemiluminescent tag. In exemplary embodiments,detecting the complex comprises carrying out surface plasmon resonanceto detect the complex or measuring change in resistance on an electrode(as FIX Padua binds to the antibody, antigen-binding fragment,polypeptide, or conjugate). See, Gonzalez-Diaz et al., “PlasmonicAu/Co/Au nanosandwiches with Enhanced Magneto-Optical Activity” Small4(2): 202-5 (2008) and Tsekenis (2008). “Label-less immunosensor assayfor myelin basic protein based upon an ac impedance protocol.”Analytical Chemistry 80 (6): 2058-62 (2008). In exemplary aspects, theenzyme is horseradish peroxidase (HRP), alkaline phosphatase (AP),glucose oxidase, or beta-galactosidase. In exemplary aspects, the enzymeis exposed to reagents which cause them to chemiluminesce or producelight. In exemplary aspects, the radioisotope is 1125. In exemplaryaspects, the DNA reporter is a DNA probe. See, e.g., Rajkovic,“Immunoquantitative real-time PCR for detection and quantification ofStaphylococcus aureus enterotoxin B in foods.” Applied and EnvironmentalMicrobiology 72 (10): 6593-9 (2006); and Gofflot “Immuno-quantitativepolymerase chain reaction for detection and quantitation of prionprotein.” Journal of Immunoassay and Immunochemistry 25 (3): 241-58(2004). In exemplary aspects, the fluorogenic reporter is phycoerythrin(PE) e.g., B-PE, R-PE, or allophycocyanin (APC).

In exemplary embodiments, the antibody or antigen-binding fragment orpolypeptide is conjugated to a detecting agent. In exemplaryembodiments, the conjugate comprises a detecting agent. In exemplaryembodiments, the antibody or antigen-binding fragment or polypeptide isnot conjugated to a detecting agent or the conjugate does not comprisesa detecting agent. In such exemplary embodiments, the methods comprisecontacting the sample with a secondary antibody comprising a detectingagent, wherein the secondary antibody binds to the antibody orantigen-binding fragment or polypeptide or conjugate. The secondaryantibody may be any antibody of any isotype or class, provided that thesecondary antibody will bind to the anti-FIX Padua antibody,antigen-binding fragment thereof, polypeptide or conjugate.

In exemplary embodiments, the antibody or antigen-binding fragment orpolypeptide is conjugated to a solid support. In exemplary embodiments,the conjugate comprises a solid support. For example, the solid supportis selected from the group consisting of a tube, a dish, a flask, a bag,a plate (e.g., a microtiter plate), a membrane, a filter, a bead, afiber, a probe, and the like. In exemplary aspects, the solid support ismade of a polymer. In exemplary aspects, the solid support is made ofagarose, cellulose, dextran, polyacrylamide, latex, or controlled poreglass. In exemplary aspects, the solid support is made of agarose. Inexemplary aspects, the solid support is made of polyvinyl difluoride(PVDF), nitrocellulose, nylon 66, protran nitrocellulose, or paper. Inexemplary aspects, the membrane is one of the Immobilon®, Proton®,QuickDraw®, Westran®, Whatman® or Hybond® membranes (Sigma-Aldrich, St.Louis, Mo.). In exemplary aspects, the solid support is a polymer bead,a magnetic or paramagnetic bead, a microtiter plate, a membrane or afilter.

Subjects

In exemplary embodiments, the subject referenced herein is a mammal,including, but not limited to, mammals of the order Rodentia, such asmice and hamsters, and mammals of the order Logomorpha, such as rabbits,mammals from the order Carnivora, including Felines (cats) and Canines(dogs), mammals from the order Artiodactyla, including Bovines (cows)and Swines (pigs) or of the order Perssodactyla, including Equines(horses). In some aspects, the mammals are of the order Primates,Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans andapes). In exemplary aspects, the mammal is a human. In exemplaryaspects, the human subject is an adult, e.g., 18 years or older, or anadolescent. In exemplary aspects, the subject has been administered anexpression vector comprising a nucleotide sequence encoding FIX Paduacomprising the amino acid sequence of SEQ ID NO: 1. In exemplaryaspects, the subject has a bleeding disorder. In exemplary aspects, thesubject has a bleeding disorder in which the subject's blood does notclot properly. In exemplary aspects, the subject has a lack ofexpression or a low expression level of Factor IX, e.g., WT Factor IX.In exemplary aspects, the subject has a mutation in the gene encodingFactor IX. In exemplary aspects, the subject suffers from hemophilia,e.g., hemophilia B (also known as Christmas Disease). In exemplaryaspects, the subject exhibits a higher than normal clotting activity. Inexemplary aspects, the subject has a naturally-occurring FIX Padua,e.g., has a gene mutation leading to expression of FIX Padua.

Samples

In exemplary embodiments, the sample referenced herein is a biologicalsample comprising one or more bodily fluids, e.g., human bodily fluids.In exemplary aspects, the sample comprises a bodily fluid, including,but not limited to, blood, plasma, serum, lymph, breast milk, saliva,mucous, semen, vaginal secretions, cellular extracts, inflammatoryfluids, cerebrospinal fluid, feces, vitreous humor, or urine obtainedfrom the subject. In exemplary aspects, the sample comprises blood,plasma, or serum. In exemplary aspects, the sample is prepared fromblood, plasma, or serum. In exemplary aspects, the sample is a fractionof blood, plasma, or serum. In exemplary aspects, the sample is a bloodsample, a plasma sample, or a serum sample. In exemplary aspects, thesample comprises blood or a fraction thereof (e.g., plasma, serum). Inexemplary aspects, the sample comprises or is plasma.

In exemplary aspects, the sample is a human tissue sample. In exemplaryaspects, the human tissue sample comprises muscle tissue, epithelialtissue, connective tissue, or nervous tissue. In exemplary aspects, thehuman tissue sample comprises bone tissue. In exemplary aspects, thehuman tissue sample comprises heart tissue, spleen tissue, lymph nodetissue, brain tissue, spinal cord tissue, nerve tissue, ear, nose or eyetissue, breast tissue, subcutaneous tissue, mammary gland tissue,myeloid tissue, lymphoid tissue, nasopharynx tissue, larynx tissue,tracheal tissue brochus tissue, lung tissue, skin tissue, salivary glandtissue, tissue from the tongue or mouth, oropharynx tissue,larngopharynx tissue, esophagus tissue, stomach tissue, small intestinetissue, appendix tissue, colon tissue, rectal tissue, anal tissue, livertissue, biliary tract tissue, pancreas tissue, gall bladder tissue,kidney tissue, ureter tissue, bladder tissue, urethra tissue, uterinetissue, vaginal tissue, vulvar tissue, ovary tissue, placenta tissue,scrotum tissue, penis tissue, prostate tissue, testicle tissue, seminalvesicle tissue, pituitary tissue, pineal tissue, thyroid tissue,parathyroid tissue, adrenal tissue, or islet of Langerhans tissue. Inexemplary aspects, the human tissue sample comprises liver tissue orspleen tissue or kidney tissue. In exemplary aspects, the human tissuesample comprises liver tissue.

The following examples are given merely to illustrate the presentinvention and not in any way to limit its scope.

EXAMPLES Example 1

This example demonstrates a method of making an antibody orantigen-binding fragment thereof of the invention.

A phage display method was used to select candidate specific FIX Paduabinding construct. The phage library was screened using a linear peptidethat enclosed the single amino acid substitution at position 338, astructural peptide that enclosed the single amino acid substitution atposition 338, or a full-length recombinant FIX Padua to capturecandidates. Three rounds of panning, with and without competition withwild-type FIX sequences, led to the identification of several candidatebinding constructs. BIACORE and ELISA experiments were performed todetermine the specificity and affinity of the candidates obtained.

One candidate (termed BC1) was obtained with a linear peptide comprisingthe sequence DRATCLLSTKFT and two candidates (termed BC2 and BC3) wereobtained with a structural peptide comprising the sequenceLVDRATCLLSTKFTIYNNMFCAGFH. Yet another candidate (BC4) was obtained witha peptide comprising a linear peptide or a structural peptidealternating with FIX Padua.

Screening assays demonstrated that the candidates bind to FIX Padua. ForBC2 and BC3 the binding signal (fold over background) to wild-type FIXwas less than 11 while the binding signal (fold over background) to FIXPadua was over 71. For BC1, the binding signal (fold over background) towild-type FIX was 1, while the binding signal to FIX Padua was nearly200. In a second set of screening assays, the binding signal towild-type FIX was 1 or less for each of BC1, BC2, and BC3, while thebinding signals for BC1, BC2, and BC3 to FIX Padua were 70, 30, and 10,respectively. BC4 demonstrated a binding signal to FIX Padua of morethan 36, while the signal to wild-type FIX was about 1.

The binding of BC1 to FIX Padua was tested via ELISA on Ni²⁺ plates inwhich coating concentrations of BC1 as well as concentrations of the FIXPadua were varied. The concentrations of BC1 and FIX Padua tested were0.04 μg/ml, 0.2 μg/ml, 1 μg/ml, and 5 μg/ml. The binding signals of BC1to FIX Padua were compared to a wt FIX control. At each concentrationtested, the binding of FIX Padua to BC1 was greater than the binding ofWT FIX to BC1 (FIG. 2).

The binding of BC1, BC2, and BC3 to FIX Padua in the presence ofwild-type FIX was tested via ELISA (as shown in FIG. 10). MaxiSorpplates were coated with 5 μg/mL of BC1, BC2, or BC3 and solutionscontaining FIX Padua at varying concentrations and 5 μg/mL wild-type FIXwere added to the coated plates. The FIX Padua concentrations testedwere 0 μg/mL, 0.156 μg/mL, 0.313 μg/mL, 0.625 μg/mL, 1.25 μg/mL, 2.5μg/mL, 5 μg/mL, and 10 μg/mL. As shown in FIG. 3, BC1 demonstrates astrong affinity for FIX Padua, even in the presence of WT Factor IX. BC1demonstrated the highest sensitivity in the presence of 5 μg/mL WTFactor IX.

The binding of BC1 to varied concentrations of FIX Padua in the presenceof 5% human plasma and 5 μg/mL WT Factor IX was tested via ELISA (asshown in FIG. 10). As shown in FIG. 4, BC1 binding to FIX Paduaincreases as the concentration of FIX Padua increases, even in thepresence of WT Factor IX and other plasma proteins. FIX Padua was variedfrom 25 ng/ml to 1000 ng/ml.

The binding of BC1 to FIX Padua in varying % plasma solutions containing50 mM benzamidine was tested via ELISA (as shown in FIG. 10). Thesolutions were 5%, 10%, or 20% (v/v) plasma. As shown in FIG. 5, BC1could detect FIX Padua in the range of 3.13 ng/ml to 200 ng/ml in the20% plasma solution.

The binding of BC1, BC2, and BC4 to FIX Padua in the presence of 20%human plasma containing 5 μg/mL WT Factor IX was tested by ELISA. Theconcentrations of FIX Padua used in this assay were 0, 3.13, 6.25, 12.5,25, 50, 100 and 200 ng/ml. As shown in FIG. 6, BC1 exhibited the highestsensitivity, while BC2 exhibited the lowest sensitivity and failed tobind to FIX Padua in the 20% human plasma sample containing 5 μg/mL WTFactor IX. BC4 demonstrated a binding ability to FIX Padua lower thanthat of BC1 (FIG. 6). Like BC2, BC3 failed to function as a FIXPadua-specific detection antibody in a 20% human plasma containingsample (data not shown).

The binding of BC1 and BC4 to other coagulation factors were tested viaELISA. In specific, the binding of BC1 or BC4 to Factor II and Factor Xwere tested. As shown in FIG. 7, the binding of BC1 was highly specificfor FIX Padua, showing no cross-reactivity against Factor II or FactorX, whereas BC4, in contrast, demonstrated a slight cross-reactivity toFactor II.

The binding of BC1 was specific for FIX Padua. In contrast, two otherbinding constructs (BC5 and BC6) were made via phase display using thestructural peptide. As shown in FIG. 8, the binding of BC5 and BC6 wasnot specific for FIX Padua, since significant binding to WT FIX wasdemonstrated.

The KD value for BC1 was determined via Biacore surface plasmonresonance. The KD of BC1 was 56 nM. BC1 demonstrated the highestaffinity for FIX Padua than the other candidates. That value is similarto, if not better than, the KD (M) values of commercial antibodies. Forexample, the KD(M) value of a commercially available sheet anti-humanwild-type FIX antibody is 3.11×10⁻⁹.

The sequences of the CDRs of the heavy and light chains of BC1 weredetermined by PCR followed by translation of the sequence and thesequences are as follows:

Heavy Chain CDR1 SEQ ID NO: 6 SSYAIS Heavy Chain CDR2 SEQ ID NO: 7GIVPAFGTANYAQKFQG Heavy Chain CDR3 SEQ ID NO: 8 SWGVISFAYLight Chain CDR1 SEQ ID NO: 9 RASQDISSYLN Light Chain CDR2 SEQ ID NO: 10AASNLQS Light Chain CDR3 SEQ ID NO: 11 MQYDSLPFTF

BC1 obtained with the linear peptide provided a unique and specific FIXPadua binding and this activity was confirmed. BC1 exhibited a detectionlimit of −3 ng/mL plasma and showed no cross reactivity to wild-type FIXeven at highly elevated (>5 μg/mL) concentrations.

These data support that a highly specific anti-FIX Padua antibody wasgenerated. This antibody binds to FIX Padua and not any of WT Factor IX,Factor II and Factor X in human plasma samples. These data support thatthis antibody can be used, e.g., for the development of clinical assaysto selectively distinguish between wild-type FIX and FIX Padua antigenlevels.

Example 2

This example demonstrates a method of using an antibody orantigen-binding fragment thereof of the invention.

The Fab fragment of a newly developed Factor IX Padua (FIXp)-specificbinding antibody (BC1) was coated on 96-well microplates at 2 μg/mLusing standard conditions. A commercially-available biotinylatedpolyclonal sheep anti-human FIX IgG and streptavidin-peroxidase wereused as a detection system. A schematic of the assay components is shownin FIG. 9.

Assay calibration was obtained by generating a six-point calibrationcurve with a FIXp preparation, covering a FIXp concentration range from27.1-0.85 ng/mL. Patients' samples were diluted with HEPES/NaCl buffercontaining 5 mg/mL bovine serum albumin, 10 mM benzamidine, 10 mM CaCl₂)and 0.05% Tween 20.

Normal human plasma or purified human FIX showed no signals in theFIXp-specific ELISA. Accurate calibration curves were obtained. FIXpspiked to 1/10-diluted normal human plasma showed acceptable recoverieswith dilution response curves parallel to that obtained for the assaystandard in buffer. Importantly, the analysis of samples of six patientstreated with an expression vector encoding FIXp demonstrated highlysimilar FIXp protein and FIX activity curves over time, and the samplesof cross-reactive material positive (CRM+) patients showed no increasedsignals for FIXp protein compared to CRM− patients, indicating thespecificity of the assay.

The FIXp-specific ELISA allows additional monitoring of treatmentoutcome by the measurement of the FIXp protein.

Example 3

This example demonstrates a selective ELISA for FIX Padua and the use ofthe ELISA for testing human plasma samples.

A preparation of BC1 (0.94 mg/mL) was diluted 1/500 with 0.1 MNaHCO₃—Na₂CO₃, pH 9.5 and bound to the wells of a Maxisorp F96 plate byincubating 100 μL/well at 0 to +10° C. overnight. The dilution buffer(DB), used for the dilution of the samples and the reagents as well asfor the blocking of the plates, was 0.1 M Hepes, 0.1 M NaCl, pH 7.2,containing 5 mg/mL biotin-free bovine serum albumin (BSA), 10 mM Ca²+,0.05% Tween 20 (Bio-Rad, EIA grade) and 10 mM benzamidine. Aftercoating, the plate was washed with phosphate-buffered saline containing0.05% Tween 20 and the wells were blocked by incubation with 200 μLDB/well at room temperature (RT, 18 to 26° C.) for 60 min. The blockingstep was terminated by washing. Then the dilutions of thestandard/samples were loaded, preparing the serial 1+1 dilution seriesdirectly on the plate. The dilutions (100 μL/well) were incubated withthe plate at RT for 60 min. Then the plate was washed again and thebiotinylated polyclonal sheep anti-human FIX detection antibody,prepared from F9-1030A (CoaChrom) was added (100 μL/well; workingdilution 1/500). After an incubation at RT for 60 min, the plate waswashed again, streptavidin peroxidase (DakoCytomation) was added (100μL/well; dilution 1/4,000) and incubated at RT for 30 min. After afinal, extensive washing procedure, bound peroxidase activity wasmeasured with the ready-to-use peroxidase substrate SureBlue, stoppingthe reaction with 3 N sulfuric acid. The plate was then measured at 450nm with an ELISA reader, subtracting the results obtained at 620 nm.

FIG. 11 shows the concentration-response curves obtained for a purifiedhuman FIX Padua preparation and a fresh-frozen control plasmapreparation (CRYOCheck; Precision Biologics) with a WT FIX concentrationof 5 μg/m L.

The dose-response curve obtained for the recombinant human FIX Paduaconcentration covering a range from 29 to 0.91 ng/mL met acceptedrequirements for accuracy, precision and linearity and was thus deemedto be appropriate for extrapolating samples. In particular, thecorrelation coefficient of the log-log regression curve was 0.9985 witha mean accuracy of 101.4% and a precision of 7.0%. Accuracy andprecision were calculated by back-fitting the signals measured for thesix concentrations of the calibration curve. The data furthermoredemonstrated the absolute specificity of the approach for FIX Padua:Human reference plasma, containing FIX wt at the normal plasmaconcentration of 5 μg/mL and measured using the minimum dilution of1/10, did not elicit any signal.

The ELISA was carried out as described above. Calibration curves wereobtained using another recombinant human FIX Padua preparation with aprotein concentration of 542.42 μg/ml. The specific clotting activity of2310 IU/mg protein clearly classified this preparation as thehyperactive FIX Padua variant. The serial dilution series ranged from1/20,000 to 1/640,000 and defined a FIX concentration range from27.1-0.85 ng/mL. FIG. 12 shows the mean calibration curve, obtained asthe linear regression curves between the logarithms of theblank-corrected mean signals and the FIX concentrations of the six assaystandards. The insert shows the agreement of the back-fitted assaycalibrators D1 top D6 with their respective nominal concentrations. Thecalibration curves for FIX Padua showed a good linearity in theconcentration range from 27.1 to 0.85 ng/mL. This was shown by the meancorrelation coefficient r=0.9992 (range: 0.9986-0.9996) and supported bythe back-fitted concentrations calculated for the individual points ofthe calibration curve, which differed by less than 9% (range: 91.1% to108.6%) from the expected ones over the whole range. These back-fittingdata easily met the requirement defined by the EMA guideline forbioanalytical method validation to identify suitable calibration curvesof ligand-binding assays. The relative total error (RTE) of thecalibration curve was low. In particular, RTE was calculated byback-fitting the mean blank-corrected optical densities (ODs) of thecalibration curve standards. The concentrations obtained were normalizedby multiplication with their dilution. The RTE was now calculated as thesum of the absolute difference between the nominal concentration of theassay standard and the mean concentration determined by the back-fittingapproach and the double standard deviation of this mean concentration.Furthermore, the low RSD of the slope demonstrated that these curvescould be obtained at a reproducibility required for an assay to be usedin a clinical setting.

The ELISA was carried out as described above. Normal fresh-frozen humanplasma and FIX-deficient plasma were spiked with FIX Padua. The dilutionseries of the two spiked plasma preparations started at the dilution of1/10. FIG. 13 shows the dilution-response curves of the two plasmasamples in comparison to those obtained in buffer. The slopes of thedilution-response curves obtained for the FIX Padua-spiked plasmasamples differed by less than 5% from that obtained for the bufferdilution series. This indicated that the plasma matrix had no influenceon the assay performance.

Conventional polyclonal anti-human FIX antibodies also bind tocynomolgus monkey FIX (data on file) due to the high sequence homologybetween human and monkey FIX. Therefore, the selectivity of the FIXPadua ELISA was checked also for the matrix of citrated monkey plasma.In particular, a female and male citrated monkey plasma sample wasspiked with FIX Padua and measured starting the dilution series at theminimum dilution of 1/10. FIG. 14 shows the dilution-response curves ofthe two plasma samples in comparison to those obtained in buffer.

The slopes of the dilution-response curves obtained for the FIXPadua-spiked plasma samples differed by less than 4% from that obtainedfor the buffer dilution series. This indicated that the citrated monkeyplasma matrix had no influence on the assay performance. Moreover, therecoveries of the spiked FIX Padua concentrations were 91.3% and 103.1%for the female and the male plasma sample, respectively.

Measurements of FIX Padua in citrated plasma samples from a subject withFIX cross-reactive material (CRM+) in a Phase 1/2 clinical trial aftertreatment with AAV2/8 viral vector expressing FIX Padua were performed.Using the plasma samples from the subject, the FIX Padua ELISA wascarried out as described above. The FIX coagulation activity and the FIXantigen measurements using the plasma samples obtained from the subjectwere performed by using standard methods. FIG. 15 shows the results ofthe FIX activity and FIX protein measurement carried out for the plasmasamples obtained from a subject from a Phase1/2 clinical trial, whichconstantly demonstrated a FIX antigen concentration of close to 1 U/mL,but FIX activity below the lower limit of quantification. This dataqualified the subject to demonstrate CRM+, i.e. coagulation-inactive FIXprotein, measurable with a conventional FIX ELISA. The results of theFIX Padua ELISA, obtained in ng/mL, were transformed to activity andantigen units, using for the activity transformation the specificactivity of the assay standard (2310 IU/mg) and for the antigentransformation a normal human FIX concentration of 5 μg/mL.

The FIX Padua ELISA data paralleled the FIX activity data, evidencingthat the FIX activity measured was dependent on the expression of FIXPadua. In contrast, the FIX antigen concentrations measured with the FIXPadua-specific ELISA were clearly lower than those obtained with thestandard ELISA, demonstrating that the FIX Padua ELISA alloweddiscrimination between CRM+ material and FIX Padua.

Measurements of FIX Padua in citrated plasma samples from a secondsubject with FIX cross-reactive material (CRM+) in a Phase 1/2 clinicaltrial after treatment with AAV2/8 viral vector expressing FIX Padua wereperformed. Using the plasma samples from the second subject, the FIXPadua ELISA was carried out as described above. The FIX coagulationactivity and the FIX antigen measurements using the plasma samplesobtained from the second subject were performed by using standardmethods. FIG. 16 shows the results of the FIX activity and FIX proteinmeasurement carried out for the plasma samples obtained from the secondsubject, which constantly demonstrated a FIX antigen concentration ofclose to 0.8 U/mL, but FIX activity below the lower limit ofquantification. This data qualified the subject to demonstrate CRM+,i.e., coagulation-inactive FIX protein, measurable with a conventionalFIX ELISA. The results of the FIX Padua ELISA, obtained in ng/mL, weretransformed to activity and antigen units, using for the activitytransformation the specific activity of the assay standard (2310 IU/mg)and for the antigen transformation a normal human FIX concentration of 5μg/mL.

The FIX Padua ELISA data paralleled the FIX activity data, evidencingthat the FIX activity measured was dependent on the expression of FIXPadua. In contrast, the FIX antigen concentrations measured with the FIXPadua-specific ELISA were clearly lower than those obtained with thestandard ELISA, demonstrating that the FIX Padua ELISA alloweddiscrimination between CRM+ material and FIX Padua.

Measurements of FIX Padua in citrated plasma samples from a subjectwithout FIX CRM+ in a Phase 1/2 clinical trial after treatment withAAV2/8 viral vector expressing FIX Padua were performed. Using theplasma samples from the subject without FIX CRM+, the FIX Padua ELISAwas carried out as described above. The FIX coagulation activity and theFIX antigen measurements using the plasma samples from the subjectwithout FIX CRM+ were performed by using standard methods. FIG. 17 showsthe results of the FIX activity and FIX protein measurement carried outfor the plasma samples obtained from the subject which constantlydemonstrated no CRM+. The results of the FIX Padua ELISA, obtained inng/mL, were transformed to activity and antigen units, using for theactivity transformation the specific activity of the assay standard(2310 IU/mg) and for the antigen transformation a normal human FIXconcentration of 5 μg/mL. The FIX Padua ELISA data paralleled the FIXactivity and FIX protein data, evidencing that the FIX activity measuredwas dependent on the expression of FIX Padua.

Example 4

A FIX chromogenic activity assay was also developed using BC1. FIX Paduawas selectively purified from the sample matrix by binding to theplate-bound BC1. Non-bound sample components, including human FIX wildtype were removed by extensive washing, before the chromogenic Factor IXTest 221806 (Hyphen Biomed) was carried out on the wells of themicroplate. The samples were diluted with the dilution buffer describedabove. FIG. 18 shows the dose response-curve obtained for FIX Padua anda normal reference plasma preparation.

FIX Padua showed a linear concentration-response curve, while the normalreference plasma concentration with a FIX concentration of about 1 U/mLdid not demonstrate any measurable signal. These data demonstrated thefeasibility of the approach, describing a FIX Padua-specific activityassay and at the same time confirmed its specificity.

Example 5

The following describes an isolation of antibodies that differentiatebetween FIX wild type and the FIX Padua variant.

FIX Padua is a naturally occurring hyper-functional variant of wild-typeFIX with a single amino acid exchange (FIX R338L). The usefulness of FIXPadua for hemophilia B gene therapy has been shown in preclinical modelsand is currently being explored in clinical phase 1/2 programs.Assessment of the success of the therapy largely relies on thedetermination of the expression of the FIX Padua transgene, which ishowever hampered by the lack of an antibody that discriminates betweenwild-type FIX and FIX Padua. Antibodies that specifically recognize FIXPadua without cross reactivity to wild-type FIX allows the developmentof assays that unambiguously detect FIX Padua in clinical samples. Aphage display method was used to select specific FIX Padua binders. Thephage library was screened with a linear and a structural peptide thatenclosed the single amino acid substitution at position 338, as well asfull-length recombinant FIX Padua. Three rounds of panning, with andwithout competition with wild-type FIX sequences, led to theidentification of several binders. BIACORE (surface plasmon resonance)and ELISA experiments were performed to determine the specificity andaffinity of the antibodies obtained. Various antibodies were initiallyidentified from the different phage display panning routes. An antibodygenerated from the linear peptide route demonstrated unique and specificFIX Padua binding. The selected antibody had a detection limit of −3ng/mL plasma and showed no cross reactivity to wild-type FIX even athighly elevated (>50 μg/mL) concentrations. The highly specific anti-FIXPadua antibody can be used for the development of clinical assays toselectively distinguish between wild-type FIX and FIX Padua antigenlevels.

INTRODUCTION

FIX Padua is a naturally occurring hyper-functional variant of FIXwild-type (wt) with a single amino acid exchange (FIX R338L). This gainin function mutation leads to an 8- to 10-fold increase in specificclotting activity compared with normal FIX. In vitro, recombinantFIX-R338L had a 5- to 10-fold higher specific clotting activity thanthat of the recombinant FIX wild type [1]. The usefulness of FIX Paduafor hemophilia B gene therapy has been shown in preclinical models, andis currently being explored in a clinical Phase 1/2 trial. Assessment ofthe success of the therapy largely relies on determination of the FIXPadua transgene expression, which is however hampered by the lack of anantibody that discriminates between FIX wt and FIX Padua.

The objective of this study was to generate antibodies that specificallybind FIX Padua without cross reactivity to FIX wild-type to allowdetection of FIX Padua in the presence of FIX wt in clinical samples.

The following describes the methods carried out during this study.

Generation of anti Padua FIX specific antibodies: Antibodies (bivalentFabs) were generated by Phage Display Technology based on HuCALPLATINUM® library and CysDisplay® technology. Four different approacheswere applied to isolate antibodies specifically for FIX Padua (FIG. 19).Two were performed with peptides that enclosed the single amino acidsubstitution at position 338 either in a solid or liquid (bead) phaseassay. The third and fourth strategy included the full-length active FIXPadua protein. Three panning rounds were performed for each strategy,including the proper negative controls (wt peptides or FIX wt). Fabs ofunique positive clones were produced and tested for specific binding toFIX Padua antigens. For ELISA, antigens [5 μg/mL] were coated andincubated with Fab fragments [2 μg/mL] followed by detection with ananti Fab AP conjugate.

Plasmatic ELISA: Fabs were coated on MaxiSorp ELISA plates [5 μg/mL] andincubated with 20% human plasma diluted in PBS buffer plus 50 mMbenzamidine. Plasma was spiked with 5 μg/mL rFIX wt and increasingconcentrations of rFIX Padua (in-house produced in HEK293). Detectionwas done using a HRP labeled polyclonal goat anti FIX antibody (100ng/mL).

BiaCore: All experiments were performed at 25° C. using a Biacore™ 200instrument and nickel-coated biosensor chips (NTA-Chip GE Healthcare).The instrument was first primed three times with HBS-EP running buffer,and flow cell 1 (FC1) was used as the reference flow cell, which wasunmodified and lacked the Fab ligand. Flow cell 2 (FC2) was used forimmobilization of ˜500 RU FIX Padua specific Ab42. Ligand concentrationsranged from 100 to 6.25 nM.

The results of this study are described below.

Structural analysis of porcine wt FIX revealed that the single Paduamodification (R338L) is located on the surface of the protein [2] (FIG.21), and is therefore a suitable epitope for the generation of highlyspecific antibodies. Purified bivalent Fabs were tested for antigenspecificity (FIG. 22) and cross reactivity to FIX wt in 20% human plasmaby ELISA (FIG. 23). In 20% plasma matrix containing 5 μg/mL spiked FIXwt, only two (Ab42 and Ab76) of four isolated bivalent Fabs boundspecifically to FIX Padua. Only Ab42 did not show any cross reactivityagainst human FII and human FX (data not shown).

Based on these data, it was concluded that a highly specific anti FIXPadua mini antibody Ab42 was generated using Phage Display technology.The selected candidate shows no cross reactivity to FIX wild type orother common blood factor proteins such as FII or FX. Effective bindingto FIX Padua was shown in a human plasma matrix (20%) and in thepresence of common concentration of FIX wt. Mini antibody Ab42 iscurrently used for FIX Padua analysis in plasma from patients treatedduring a clinical Phase 1/2 trial (see below).

In the study described above, the following references are cited: (1)Simioni et al., X-Linked Thrombophilia with a Mutant Factor IX (FactorIX Padua) N Engl J Med 2009; 361: 1671-5; and (2) Brandstetter et al.,X-ray structure of dotting factor IXa: active site and module structurerelated to Xase activity and hemophilia B. Proc Nail Acad Sci USA. 1995Oct. 10; 92(21): 9796-800

The following describes a development and application of a FIXPadua-specific immunoassay for the monitoring of hemophilia B genetherapy.

Gene therapy holds great promise as a future treatment option forHemophilia. In one clinical Phase 1/2 trial, an AAV2/8 viral vector isused to express FIX Padua (FIXp), a hyper-functional variant of FIX witha single amino acid exchange (R338L), in subjects with severe hemophiliaB. Specific detection of the transgene product is key for assessing thesuccess of the therapy, but challenging for patients with FIXcross-reactive material (CRM+). Development of a FIXp-specific ELISA andapplication of this assay for the measurement of FIXp expressed in theplasma of hemophilia B patients after treatment with AAV2/8 viral vectorexpressing FIX Padua. The Fab fragment of a newly developedFIXp-specific binding antibody was coated to 96-well microplates at 2μg/mL using standard conditions. A biotinylated polyclonal sheepanti-human FIX IgG and streptavidin peroxidase were used as detectionsystem. Assay calibration was obtained by generating a six-pointcalibration curve with a FIXp preparation, covering a FIXp concentrationrange from 27.1-0.85 ng/mL. Patients' samples were diluted withHEPES/NaCl buffer containing 5 mg/mL bovine serum albumin, 10 mMbenzamidine, 10 mM CaCl₂) and 0.05% Tween 20. Normal human plasma orpurified human FIX showed no signals in the FIXp-specific ELISA.Accurate calibration curves were obtained. FIXp spiked to 1/10-dilutednormal human plasma showed acceptable recoveries with dilution responsecurves parallel to that obtained for the assay standard in buffer.Importantly, the analysis of samples of six patients treated with AAVencoding FIXp demonstrated highly similar FIXp protein and FIX activitycurves over time, and the samples of CRM+ patients showed no increasedsignals for FIXp protein compared to CRM− patients, indicating thespecificity of the assay. The FIXp-specific ELISA allows additionalmonitoring of treatment outcome by the measurement of the FIXp protein.This represents the first data demonstrating the feasibility of thisapproach.

INTRODUCTION

Gene therapy holds great promise as a future treatment option forhemophilia [1]. In a clinical Phase 1/2 trial, an AAV2/8 viral vector isused to express FIX Padua (FIXp) [2], a hyper-functional variant of FIXwith a single amino acid exchange (R338L), in subjects with severehemophilia B. Specific detection of the transgene product is crucial forassessing the success of the therapy, but challenging for patients withFIX cross-reactive material (CRM+).

FIG. 25 depicts the principle of the method (assay) described in thisstudy. Anti-FIX Padua Fab is coated to microplate wells and selectivelycaptures FIX Padua from the sample. After a washing step, removingnon-bound sample compounds, the bound FIX Padua is detected using anin-house biotinylated polyclonal sheep anti-FIX IgG and streptavidinperoxidase. The bound HRP activity is measured using the ready-to-useHRP substrate SureBlue.

The following describes the methods carried out during this study.

ELISA procedure: The Fab preparation Ab42 (0.94 mg/mL) was diluted 1/500with 0.1 M NaHCO₃—Na₂CO₃, pH 9.5 and bound to the wells of Maxisorp F96plate by incubating 100 μL/well at 0 to +10° C. overnight. The dilutionbuffer (DB), used for dilution of the samples and reagents and forblocking of the plates, contained 0.1 M HEPES, 0.1 M NaCl, pH 7.2, 5mg/mL biotin-free bovine serum albumin (BSA), 10 mM Ca²+, 0.05% Tween 20(Bio-Rad, EIA grade), and 10 mM benzamidine. After coating, the platewas washed with phosphate-buffered saline containing 0.05% Tween 20. Thewells were then blocked by incubation with 200 μL DB/well at roomtemperature (RT) for 60 min. The blocking step was terminated bywashing. Then, the dilutions of the standard/samples were loaded,preparing the serial 1+1 dilution series directly on the plate. Thedilutions (100 μL/well) were incubated at RT for 60 min. The plate waswashed again, and the biotinylated polyclonal sheep anti-human FIXdetection antibody prepared from F9-1030A (A-Coa) added (100 μL/well;dilution 11500). After incubation at RT for 60 min, the plate was washedagain, and streptavidin peroxidase (DakoCytomation) added (100 μL/well:dilution 1/4,000) and incubated at RT for 30 min. After a final washingprocedure, the bound peroxidase activity was measured with theready-to-use peroxidase substrate SureBlue (KPL), stopping the reactionwith 3 N sulfuric acid. The plate was then measured at 450 nm,subtracting the results obtained at 620 nm. The calibration curve wasconstructed with a purified recombinant FIX Padua (FIXp) preparation,demonstrating a protein concentration of 542.4 μg/mL. The specificclotting activity of 2,310 IU/mg protein clearly classified thispreparation as the hyperactive FIX Padua variant. The serial dilutionseries ranged from 1/20,000 to 1/640,000 and defined a FIX concentrationrange from 27.1 to 0.85 ng/m L.

The results of this study are described below and in the figures. TheFIX coagulation activity and the FIX antigen measurements were performedapplying established standard methods; the citrated plasma samples werealso subjected to specific measurement of FIX Padua protein with ELISA.The results of the FIX Padua ELISA, obtained in ng/mL, were transformedto activity and antigen units. In particular, the specific activity ofthe recombinant purified FIX Padua preparation of 2,310 IU FIX/mg,applied as assay standard for FIXp ELISA, was used to calculate activityunits, while the transformation to antigen plasma units was based on thenormal FIX plasma concentration of 5 μg/m L. FIGS. 26-29 demonstrate theassay selectivity, calibration curves, parallelism study in normal andFIX-deficient plasma and the influence of calcium on the sensitivity ofELISA, respectively

Plasma samples were obtained from patients treated with an AAV2/8 viralvector. FIGS. 30-32 demonstrate activity and expression of FIX Padua insamples obtained from three patients. The plasma samples obtained fromSubject 05-001 consistently demonstrated a FIX antigen concentration ofclose to 1 U/mL, but FIX activity below the lower limit ofquantification. This data revealed the presence of FIX cross-reactivematerial (CRM+), i.e. coagulation-inactive FIX protein, measurable witha conventional FIX ELISA. The FIXp ELISA data paralleled the FIXactivity data, demonstrating that the FIX activity measured wasdependent on the expression of FIX Padua. In contrast, the FIX antigenconcentrations measured with the FIX Padua-specific ELISA were clearlylower than those obtained with the standard ELISA, demonstrating thatthe FIX Padua ELISA allowed discrimination between CRM+ material and FIXPadua. Similar data, although at lower CRM+ levels, were found for thesamples from Subject 10-005, while those from Subject 03-004 did notcontain CRM+, resulting in parallel time-versus-concentration curvesalso for the two ELISA systems.

From these data, it was concluded that the FIX Padua-specific ELISA,based on using the highly specific Fab fragment for the capture of FIXp,allows additional monitoring of treatment outcome by the measurement ofFIXp. These are the first data demonstrating the feasibility of thisapproach.

In the study described above, the following references are cited: [1]Simioni P, et al (2009): NEJM 361, 1671-1675. X-Linked thrombophiliawith a Mutant Factor IX (Factor IX Padua); [2] Crudele J M, et al(2015). Blood 125:1553-1561. AAV liver expression of FIX-Padua preventsand eradicates FIX inhibitor without increasing thrombogenicity inhemophilia B dogs and mice.

Embodiments of the present specification may also be described asfollows:

-   1. An antibody or antigen-binding fragment thereof that binds a    Factor IX Padua comprising the amino acid sequence of SEQ ID NO: 1    and does not bind to a wild-type (WT) Factor IX comprising the amino    acid sequence (SEQ ID NO: 2).-   2. The antibody or antigen-binding fragment of embodiment 1, which    binds an epitope of SEQ ID NO: 1, wherein the epitope is a linear    epitope within the amino acid sequence DRATCLLSTKFT (SEQ ID NO: 3).-   3. The antibody or antigen-binding fragment of embodiment 1, which    binds to an epitope of SEQ ID NO: 1, wherein the epitope is a    conformational epitope of the folded structure of the amino acid    sequence LVDRATCLLSTKFTIYNNMFCAGFH (SEQ ID NO: 5).-   4. The antibody or antigen-binding fragment of embodiment 1 or 3,    wherein the folded structure comprises a disulfide bridge.-   5. The antibody or antigen-binding fragment of any one of the    previous embodiments, which does not bind to the amino acid sequence    of DRATCLRSTKFT (SEQ ID NO: 14) or LVDRATCLRSTKFTIYNNMFCAGFH (SEQ ID    NO:15).-   6. The antibody or antigen-binding fragment of any one of the    previous embodiments, wherein the antibody or antigen-binding    fragment binds to the Factor IX Padua with a K_(D) of about 100 nM    or less.-   7. The antibody or antigen-binding fragment of any one of the    previous embodiments, wherein the antibody or antigen-binding    fragment binds to the Factor IX Padua with a K_(D) within a range of    about 25 to about 75 nM.-   8. The antibody or antigen-binding fragment of any one of the    previous embodiments, wherein the antibody or antigen-binding    fragment binds to the Factor IX Padua with a K_(D) within a range of    about 50 nM to about 60 nM.-   9. The antibody or antigen-binding fragment of any one of the    previous embodiments, wherein the antibody or antigen-binding    fragment binds to the Factor IX Padua with a K_(D) within a range of    about 20 nM to about 100 nM, about 25 nM to about 95 nM, about 30 nM    to about 90 nM, about 35 nM to about 85 nM, about 40 nM to about 80    nM, about 45 nM to about 75 nM, about 50 nM to about 70 nM, or about    55 nM to about 65 nM.-   10. The antibody or antigen-binding fragment of any one of the    previous embodiments, wherein the antibody or antigen-binding    fragment binds to the Factor IX Padua and does not bind to a WT    Factor IX in a sample comprising human plasma, optionally, wherein    the sample comprises at least or about 5%, at least or about 10%, or    at least or about 20% human plasma and the sample comprises at least    or about 5 μg/mL WT Factor IX.-   11. The antibody or antigen-binding fragment of any one of the    previous embodiments, wherein the antibody or antigen-binding    fragment does not bind to a Factor II polypeptide or a Factor X    polypeptide.-   12. The antibody or antigen-binding fragment of any one of the    previous embodiments, wherein the antibody or antigen-binding    fragment binds to neither Factor II nor Factor X.-   13. The antibody or antigen-binding fragment of any one of the    previous embodiments, which is a Fab or Fab2′ antibody fragment.-   14. The antibody or antigen-binding fragment of any one of the    previous embodiments, which is monospecific.-   15. The antibody or antigen-binding fragment of any one of the    previous embodiments, which is fully human.-   16. The antibody or antigen-binding fragment of any one of the    previous embodiments, which is bivalent.-   17. The antibody or antigen-binding fragment of any one of the    previous embodiments, which is bivalent but monospecific for FIX    Padua.-   18. The antibody or antigen-binding fragment of any one of the    previous embodiments, comprising dimerized Fab fragments or    dimerized Fab mini antibody.-   19. The antibody or antigen-binding fragment of any one of the    previous embodiments, which is a dimerized Fab fragment via a    linker.-   20. The antibody or antigen-binding fragment of any one of the    previous embodiments, comprising (i) the amino acid sequences of:    SSYAIS (SEQ ID NO: 6); GIVPAFGTANYAQKFQG (SEQ ID NO: 7); SWGVISFAY    (SEQ ID NO: 8); RASQDISSYLN (SEQ ID NO: 9); AASNLQS (SEQ ID NO: 10);    and MQYDSLPFTF (SEQ ID NO: 11) or (ii) the amino acid sequence of    SEQ ID NO: 24 or SEQ ID NO: 25 or SEQ ID NOs: 24 and 25 or (iii) the    amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 27 or SEQ ID NOs:    26 and 27.-   21. A polypeptide comprising an amino acid sequence comprising each    of SEQ ID NOs: 6-11, optionally, wherein (i) one or more amino acids    are present between each of SEQ ID NOs: 6-11, and/or (ii) the    polypeptide optionally further comprises a FLAG tag comprising    DYKDDDDK (SEQ ID NO: 12) and/or a hexa-His tag comprising HHHHHH    (SEQ ID NO: 13).-   22. The polypeptide of embodiment 21, wherein the FLAG tag and/or    the hexa-His tag are located at the C-terminal end of the    polypeptide.-   23. A conjugate comprising the antibody or antigen-binding fragment    or polypeptide of any one of the previous embodiments, conjugated to    a heterologous moiety.-   24. A conjugate of embodiment 23, wherein the heterologous moiety    can be selected from the group consisting of: a polymer, a    carbohydrate, a lipid, a nucleic acid, an oligonucleotide, an amino    acid, peptide, polypeptide, protein and a detecting agent.-   25. The conjugate of embodiment 23 or 24, wherein the antibody or    antigen-binding fragment or polypeptide is conjugated to agarose,    cellulose, dextran, polyacrylamide, latex or controlled pore glass.-   26. The conjugate of any one of embodiments 23 to 25, wherein the    antibody or antigen-binding fragment or polypeptide is conjugated to    a fluorophore, chromophore, radioisotope, enzymatic label, or    biotin.-   27. The conjugate of any one of embodiments 23 to 26, comprising a    homodimer of the polypeptide of embodiment 19.-   28. The conjugate of any one of embodiment 27, wherein the    polypeptides of the dimer are linked via a helix-turn-helix    structure.-   29. A nucleic acid comprising a nucleotide sequence encoding the    antibody, antigen-binding fragment, polypeptide, conjugate, or a    fragment thereof, of any one of the previous embodiments.-   30. A vector comprising the nucleic acid of embodiment 29.-   31. A host cell comprising the nucleic acid of embodiment 29 or the    vector of embodiment 30.-   32. A kit comprising the antibody or antigen-binding fragment of any    one of embodiments 1 to 20, the polypeptide of embodiment 21 or 22,    the conjugate of any one of embodiments 23 to 28, the nucleic acid    of embodiment 29, the vector of embodiment 30, and/or the host cell    of embodiment 31.-   33. The kit of embodiment 32, further comprising a secondary    antibody which binds to the antibody, antigen-binding fragment,    polypeptide, or conjugate.-   34. The kit of embodiment 32 or 33, further comprising a solid    support.-   35. The kit of any one of embodiments 32 to 34, wherein the    antibody, antigen-binding fragment, polypeptide or conjugate is    pre-coated on a solid support.-   36. The kit of embodiment 34 or 35, wherein the solid support is a    polymer bead, a microtiter plate, a membrane, or a filter.-   37. The kit of embodiment 35 or 36, comprising a solid support    pre-coated with a solution comprising about 100 ng or more, about    150 ng or more, about 200 ng or more, about 500 ng or more of the    antigen binding fragment.-   38. The kit of any one of embodiments 32 to 37, wherein the kit    further comprises instructions for use.-   39. A composition comprising an antibody or antigen-binding fragment    thereof according to any one of embodiments 1 to 20 admixed with a    biological sample obtained from a human comprising human plasma, or    a diluted fraction thereof, and/or human tissue, or cells thereof,    wherein, optionally, the composition comprises a detecting agent.-   40. A composition comprising an antibody or antigen-binding fragment    thereof according to any one of embodiments 1 to 20 admixed with a    biological sample obtained from a human comprising human plasma    proteins, wherein at least one of the human plasma proteins is    selected from the group consisting of Factor IX, a variant of Factor    IX, Factor II, a variant of Factor II, Factor X, and a variant of    Factor X.-   41. A composition of embodiment 40, wherein the composition    comprises a detecting agent.-   42. Use of the antibody or antigen-binding fragment of any one of    embodiments 1 to 20, the polypeptide of embodiment 21 or 22, the    conjugate of any one of embodiments-   23 to 28, the nucleic acid of embodiment 29, the vector of    embodiment 30, the host cell of embodiment 31, and/or the kit of any    one of embodiments 32 to 38 for detecting Factor IX Padua in a    sample.-   43. A method of detecting Factor IX Padua in a sample obtained from    a subject, comprising (i) contacting the sample with the antibody or    antigen-binding fragment of any one of embodiments 1 to 20, the    polypeptide of embodiment 21 or 22, or the conjugate of any one of    embodiments 23 to 28 to form a complex comprising the Factor IX    Padua and the antibody, antigen-binding fragment, polypeptide or    conjugate, and (ii) detecting the complex in the sample.-   44. The method of embodiment 43, wherein the antibody or    antigen-binding fragment or polypeptide is conjugated to a detecting    agent and/or a solid support or wherein the conjugate comprises a    detecting agent.-   45. The method of embodiment 43 or 44, comprising contacting the    sample with a secondary antibody comprising a detecting agent,    wherein the secondary antibody binds to the antibody or    antigen-binding fragment or polypeptide or conjugate.-   46. The method of any one of embodiments 43 to 35, wherein detecting    the complex comprises detecting a signal of the detecting agent.-   47. The method of embodiment 46, wherein the signal is an enzymatic    activity, binding activity and/or chromogenic activity.-   48. The method of any one of embodiments 43 to 47, wherein the    sample is a blood sample, a serum sample, or a plasma sample.-   49. The method of any one of embodiments 43 to 48, wherein the    subject has been treated with a vector comprising a nucleotide    sequence encoding Factor IX Padua.-   50. A binding construct described herein.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the disclosure (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range and each endpoint, unless otherwise indicatedherein, and each separate value and endpoint is incorporated into thespecification as if it were individually recited herein.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to better illuminate thedisclosure and does not pose a limitation on the scope of the disclosureunless otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element as essential to thepractice of the disclosure.

Preferred embodiments of this disclosure are described herein, includingthe best mode known to the inventors for carrying out the disclosure.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the disclosure to be practicedotherwise than as specifically described herein. Accordingly, thisdisclosure includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the disclosure unlessotherwise indicated herein or otherwise clearly contradicted by context.

What is claimed is:
 1. An antibody or antigen-binding fragment thereofthat binds a Factor IX Padua comprising the amino acid sequence of SEQID NO: 1 and does not bind to a wild-type (WT) Factor IX comprising theamino acid sequence (SEQ ID NO: 2), wherein the antibody orantigen-binding fragment thereof comprises: (i) the amino acid sequencesof: SSYAIS (SEQ ID NO: 6), GIVPAFGTANYAQKFQG (SEQ ID NO: 7), SWGVISFAY(SEQ ID NO: 8), RASQDISSYLN (SEQ ID NO: 9), AASNLQS (SEQ ID NO: 10), andMQYDSLPFTF (SEQ ID NO: 11); or (ii) the amino acid sequences of SEQ IDNOs: 24 and 25; or (iii) the amino acid sequences of SEQ ID NOs: 26 and27.
 2. The antibody or antigen-binding fragment of claim 1, which bindsan epitope of SEQ ID NO: 1, wherein the epitope is a linear epitopewithin the amino acid sequence DRATCLLSTKFT (SEQ ID NO: 3).
 3. Theantibody or antigen-binding fragment of claim 1, which binds to anepitope of SEQ ID NO: 1, wherein the epitope is a conformational epitopeof the folded structure of the amino acid sequenceLVDRATCLLSTKFTIYNNMFCAGFH (SEQ ID NO: 5), optionally, wherein the foldedstructure comprises a disulfide bridge.
 4. The antibody orantigen-binding fragment of claim 1, which does not bind to the aminoacid sequence of DRATCLRSTKFT (SEQ ID NO: 14) orLVDRATCLRSTKFTIYNNMFCAGFH (SEQ ID NO:15).
 5. The antibody orantigen-binding fragment of claim 1, wherein the antibody orantigen-binding fragment binds to the Factor IX Padua with a K_(D) ofabout 100 nM or less.
 6. The antibody or antigen-binding fragment ofclaim 5, wherein the antibody or antigen-binding fragment binds to theFactor IX Padua with a K_(D) within a range of about 25 to about 75 nM,optionally, wherein the antibody or antigen-binding fragment binds tothe Factor IX Padua with a K_(D) within a range of about 50 nM to about60 nM.
 7. The antibody or antigen-binding fragment of claim 1, whereinthe antibody or antigen-binding fragment binds to the Factor IX Paduaand does not bind to a WT Factor IX in a sample comprising human plasma,optionally, wherein the sample comprises at least or about 5%, at leastor about 10%, or at least or about 20% human plasma and the samplecomprises at least or about 5 μg/mL WT Factor IX.
 8. The antibody orantigen-binding fragment of claim 1, wherein the antibody orantigen-binding fragment does not bind to a Factor II polypeptide or aFactor X polypeptide, optionally, wherein the antibody orantigen-binding fragment binds to neither Factor II nor Factor X.
 9. Theantibody or antigen-binding fragment of claim 1, which is a Fab or Fab2′antibody fragment.
 10. The antibody or antigen-binding fragment of claim1, which is monospecific.
 11. The antibody or antigen-binding fragmentof claim 1, which is fully human.
 12. The antibody or antigen-bindingfragment of claim 1, which is bivalent.
 13. The antibody orantigen-binding fragment of claim 1, comprising dimerized Fab fragmentsor dimerized Fab mini antibody.
 14. The antibody or antigen-bindingfragment of claim 1, which is a dimerized Fab fragment via a linker. 15.An antibody or antigen-binding fragment comprising an amino acidsequence comprising each of SEQ ID NOs: 6-11, wherein the antibody orantigen-binding fragment binds a Factor IX Padua comprising the aminoacid sequence of SEQ ID NO: 1 and does not bind to a wild-type (WT)Factor IX comprising the amino acid sequence (SEQ ID NO: 2), andoptionally, wherein the antibody or antigen-binding fragment furthercomprises a FLAG tag comprising DYKDDDDK (SEQ ID NO: 12) and/or ahexa-His tag comprising HHHHHH (SEQ ID NO: 13), optionally, wherein theFLAG tag and/or the hexa-His tag are located at the C-terminal end ofthe antibody or antigen-binding fragment.
 16. A conjugate comprising theantibody or antigen-binding fragment of claim 1, conjugated to aheterologous moiety, optionally selected from the group consisting of: apolymer, a carbohydrate, a lipid, a nucleic acid, an oligonucleotide, anamino acid, peptide, polypeptide, protein and a detecting agent.
 17. Theconjugate of claim 16, wherein the antibody or antigen-binding fragmentis conjugated to agarose, cellulose, dextran, polyacrylamide, latex orcontrolled pore glass.
 18. The conjugate of claim 16, wherein theantibody or antigen-binding fragment is conjugated to a fluorophore,chromophore, radioisotope, enzymatic label, or biotin.
 19. The conjugateof claim 16, comprising a homodimer, which is a dimerized Fab fragmentvia a linker, optionally, wherein the polypeptides of the dimer arelinked via a helix-turn-helix structure.
 20. A nucleic acid comprising anucleotide sequence encoding the antibody or antigen-binding fragment ofclaim
 1. 21. A vector comprising the nucleic acid of claim
 20. 22. Ahost cell comprising the nucleic acid of claim
 20. 23. A kit comprising(i) the antibody or antigen-binding fragment of claim 1, and optionally,instructions for use, and optionally, (ii) a secondary antibody whichbinds to the antibody or antigen-binding fragment of (i).
 24. The kit ofclaim 23, further comprising a solid support.
 25. The kit of claim 24,wherein the antibody or antigen-binding fragment is pre-coated on thesolid support.
 26. The kit of claim 24, wherein the solid support is apolymer bead, a microtiter plate, a membrane, or a filter.
 27. The kitof claim 26, comprising a solid support pre-coated with a solutioncomprising about 50 ng to about 500 ng of the antibody or antigenbinding fragment.
 28. A composition comprising an antibody orantigen-binding fragment of claim 1 admixed with a biological sampleobtained from a human comprising human plasma, or a diluted fractionthereof, and/or human tissue, or cells thereof, wherein, optionally, thecomposition comprises a detecting agent.
 29. A composition comprising anantibody or antigen-binding fragment of claim 1 admixed with abiological sample obtained from a human comprising human plasmaproteins, wherein at least one of the human plasma proteins is selectedfrom the group consisting of Factor IX, or a variant thereof, Factor II,and Factor X, wherein, optionally, the composition comprises a detectingagent.
 30. A method of detecting Factor IX Padua in a sample obtainedfrom a subject, comprising (i) contacting the sample with the antibodyor antigen-binding fragment of claim 1 to form a complex comprising theFactor IX Padua and the antibody or antigen-binding fragment, and (ii)detecting the complex in the sample.
 31. The method of claim 30, whereinthe antibody or antigen-binding fragment is conjugated to a detectingagent and/or a solid support.
 32. The method of claim 30, comprisingcontacting the sample with a secondary antibody comprising a detectingagent, wherein the secondary antibody binds to the antibody orantigen-binding fragment.
 33. The method of claim 31, wherein detectingthe complex comprises detecting a signal of the detecting agent.
 34. Themethod of claim 33, wherein the signal is an enzymatic activity, bindingactivity and/or chromogenic activity.
 35. The method of claim 30,wherein the sample is a blood sample, a serum sample, or a plasmasample.
 36. The method of claim 30, wherein the subject has been treatedwith a vector comprising a nucleotide sequence encoding Factor IX Padua.37. A method of detecting Factor IX Padua in a sample obtained from asubject, comprising (i) contacting the sample with the antibody orantigen-binding fragment of claim 15 to form a complex comprising theFactor IX Padua and the antibody or antigen-binding fragment, and (ii)detecting the complex in the sample.
 38. The method of claim 33, whereinthe antibody or antigen-binding fragment is conjugated to a detectingagent and/or a solid support.
 39. The method of claim 33, comprisingcontacting the sample with a secondary antibody comprising a detectingagent, wherein the secondary antibody binds to the antibody orantigen-binding fragment.
 40. The method of claim 38, wherein detectingthe complex comprises detecting a signal of the detecting agent.
 41. Themethod of claim 40, wherein the signal is an enzymatic activity, bindingactivity and/or chromogenic activity.
 42. The method of claim 33,wherein the sample is a blood sample, a serum sample, or a plasmasample.
 43. The method of claim 33, wherein the subject has been treatedwith a vector comprising a nucleotide sequence encoding Factor IX Padua.44. A method of detecting Factor IX Padua in a sample obtained from asubject, comprising (i) contacting the sample with the conjugate ofclaim 16 to form a complex comprising the Factor IX Padua and theconjugate, and (ii) detecting the complex in the sample.
 45. The methodof claim 44, wherein the conjugate comprises a detecting agent.
 46. Themethod of claim 44, comprising contacting the sample with a secondaryantibody comprising a detecting agent, wherein the secondary antibodybinds to the conjugate.
 47. The method of claim 45, wherein detectingthe complex comprises detecting a signal of the detecting agent.
 48. Themethod of claim 47, wherein the signal is an enzymatic activity, bindingactivity and/or chromogenic activity.
 49. The method of claim 44,wherein the sample is a blood sample, a serum sample, or a plasmasample.
 50. The method of claim 44, wherein the subject has been treatedwith a vector comprising a nucleotide sequence encoding Factor IX Padua.51. A kit comprising (i) the antibody or antigen-binding fragment ofclaim 15, and optionally, instructions for use, and optionally, (ii) asecondary antibody which binds to the antibody or antigen-bindingfragment of (i).
 52. A kit comprising (i) the conjugate of claim 16, andoptionally, instructions for use, and optionally, (ii) a secondaryantibody which binds to the conjugate of (i).